Detection of sarcocystis neurona

ABSTRACT

A gene encoding a 29 kilodalton protein found on the surface of merozoite stage  S. neurona  has been cloned and sequenced. The protein encoded by this gene, termed SnSAG-1, is an immunodominant antigen recognized on protein blots. Methods for using nucleic acids and polypeptides relating to SnSAG-1 in diagnostic tests and vaccine development are disclosed.

CROSS REFERENCE TO RELATED APPLICATION

The present application claims the priority of U.S. provisional patentapplication No. 60/234,676 filed Sep. 22, 2000.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with United States Government support undergrant number 98-35204-6487 awarded by the United States Department ofAgriculture. The U.S. government may have certain rights in theinvention.

FIELD OF THE INVENTION

The invention relates generally to the fields of microbiology andveterinary medicine. More particularly, the invention concernscompositions and methods relating to detecting Sarcocystis neurona.

BACKGROUND

Equine Protozoal Myeloencephalitis (EPM) is a common cause of neurologicdisease in New World horses. It is caused by a parasite termedSarcocystis neurona (S. neurona), an obligatory intracellularapicomplexan parasite whose multi-phase life cycle is completed ineither one or two hosts. S. neurona is known to cycle naturally betweenopossums and both none-banded armadillos and striped skunks. Horsestypically become infected by consuming infectious parasite stages foundin opossum feces. Once a horse has been infected, S. neurona can travelto the brain and spinal cord, where merozoite stages of this parasitereplicate and cause pathology.

Horses with EPM typically present with lameness, but may alternativelyor additionally present with symptoms characteristic of primary braindisease. Because the parasite can inhabit any area of the centralnervous system (CNS) of the horse, symptoms associated with EPM can varywidely. The degree of infection can range from subtle to severe and caninvolve the brain and/or the spinal cord. EPM is usually progressive.

Presently, a definitive diagnosis of EPM is made by post-mortemexamination, where S. neurona organisms are identified in histologicallesions. The organ may also be cultured from the lesion. The presence ofthe organism in the histologic section or when cultured from the lesionestablishes the diagnosis. Heretofore, pre-mortem methods for diagnosingEPM were based on assays using whole merozoites, and not a purifiedprotein, to probe for the presence of anti-S. neurona antibodies (as anindication of infection) in the horses. The use of such whole merozoitesresults in significant cross-reaction with non-S. neurona specificantibodies (e.g., those against other Sarcocystis species). Thiscross-reactivity obscures interpretation of results using wholemerozoite-based assays.

SUMMARY

The invention relates to the discovery and characterization of a 29kilodalton (kDa) protein found on the surface of merozoite stage S.neurona. This antigen, termed SnSAG-1 or SnSMA1, is an immunodominantantigen recognized on protein blots. Using purified or recombinantSnSAG-1 (i.e., rSnSAG-1) antigen, accurate assays for diagnosing EPM inhorse pre-mortem have been developed. These assays involve identifying amarker indicative of the presence of the 29 kDa antigen or an antibodyto this antigen in a sample to be tested. Thus, because a singlepurified antigen or marker is utilized in such assays, thecross-reactivity problems associated with whole-merozite based assaysare obviated or much reduced.

A cDNA copy of the mRNA which encodes the SnSAG-1 antigen has beencloned from a gene library prepared from an isolate of S. neurona. Theoriginal clone was identified in a collection of random sequence tagsprepared to characterize the cDNA library. Additional clones of the samegene sequence were obtained to identify a full length gene. Thenucleotide sequence of a full-length gene clone was determined. Thissequence or the clone itself can be used to gusto prepare the SnSAG-1antigen in a recombinant or other synthetic form for use in diagnostictests and vaccine development.

Accordingly, the invention features a composition for detecting thepresence of S. neurona in a biological sample. In one variation, thecomposition includes a SnSAG-1 marker that is a purified nucleic acidincluding a nucleotide sequence that encodes a protein that shares atleast 50% or at least 90% sequence identity with SEQ ID NO:1. In thisvariation, the nucleotide sequence can also encode the protein of SEQ IDNO:1. For example, the nucleotide sequence can be SEQ ID NO:3.

In a second variation of the composition, the SnSAG-1 marker is apurified polynucleotide that binds under stringent hybridizationconditions to a complement of the nucleotide sequence SEQ ID NO:3,wherein the polynucleotide is at least 30 (e.g., 50, 100, or 200)nucleotides in length.

In a third variation of the composition, the SnSAG-1 marker is anisolated protein including a polypeptide that shares at least 50%, 70%,90%, or 95% sequence identity with a fragment of the amino acid sequenceof SEQ ID NO:1 that is at least 20, 50, 100, or 300 contiguous residuesin length. For example, the polypeptide can include at least 20, 50,100, or 300 contiguous amino acid residues of the sequence of SEQ IDNO:1. The polypeptide can also include the entire amino acid sequence ofSEQ ID NO:1. In this composition, the protein can be a fusion protein ora recombinant protein.

In a fourth variation of the composition, the SnSAG-1 marker is apurified antibody that specifically binds to a polypeptide consisting ofthe amino acid sequence of SEQ ID NO:1, wherein the antibody is amonoclonal antibody or a monospecific polyconal antibody. The purifiedantibody can be labeled with a detectable label such as a radioisotope,a fluorescent compound, a bioluminescent compound, a chemiluminescentcompound, biotin, colloidal gold, a magnetic particle, or an enzyme.

In another aspect, the invention features a method for detectingSarcocystis neurona in a biological sample. This method includes thesteps of: (a) providing the biological sample; and (b) analyzing thebiological sample for the presence of a SnSAG-1 marker that is a nucleicacid including a nucleotide sequence that encodes a protein that sharesat least 50% sequence identity with SEQ ID NO:1; a polynucleotide thatbinds under stringent hybridization conditions to a complement of thenucleotide sequence SEQ ID NO:3, wherein the polynucleotide can be atleast 30 nucleotides in length; a protein including a polypeptide thatshares at least 50% sequence identity with a fragment of the amino acidsequence of SEQ ID NO:1 that can be at least 20 contiguous residues inlength; or an antibody that specifically binds to a polypeptideconsisting of the amino acid sequence of SEQ ID NO:1. In this method,the presence of the SnSAG-1 marker in the biological sample indicatesthat the biological sample contains Sarcocystis neurona.

In the variation of this method where the SnSAG-1 marker is a nucleicacid including a nucleotide sequence that encodes a protein that sharesat least 50% sequence identity with SEQ ID NO:1, the nucleotide sequencecan be SEQ ID NO:3.

In the variation of this method where the SnSAG-1 marker is a proteinincluding a polypeptide that shares at least 50% sequence identity witha fragment of the amino acid sequence of SEQ ID NO:1 at least 20contiguous residues in length, the polypeptide can include at least 20contiguous amino acid residues of the sequence of SEQ ID NO:1. Forexample, the polypeptide can include the amino acid sequence of SEQ IDNO:1.

The biological sample of the method can include CNS tissue, CSF, blood,or serum. It can also be derived from a horse.

In this method of the inevtnion, the step (B) of analyzing thebiological sample for the presence of a SnSAG-1 marker can includeisolating RNA from the sample, generating cDNAs from the isolated RNA,and amplifying the cDNAs by PCR to generate a PCR product. The step (B)of analyzing the biological sample for the presence of a SnSAG-1 markercan also include contacting the sample with a labeled oligonucleotideprobe that hybridizes under stringent hybridization conditions to thenucleotide sequence SEQ ID NO:3 or a complement of the nucleotidesequence SEQ ID NO:3; or contacting the sample to a molecule thatspecifically binds to an antibody that specifically binds a proteinconsisting of the amino acid sequence of SEQ ID NO:1. In the latter, themolecule can be immobilized on a substrate.

In one variation of the method, the biological sample includes SnSAG-1specific antibodies that are specifically bound to the moleculeimmobilized on substrate. The antibodies can be detected using asecondary antibody that is labeled with a detectable label. For example,the secondary antibody can specifically bind horse immunoglobulin. Thedetectable label can be a radioisotope, a fluorescent compound, abioluminescent compound, a chemiluminescent compound, biotin, colloidalgold, a magnetic particle, or an enzyme (e.g., peroxidase or alkalinephosphatase).

In another variation of this method, the step (B) of analyzing thebiological sample for the presence of a SnSAG-1 marker includescontacting the sample with a molecule that specifically binds a proteinconsisting of the amino acid sequence of SEQ ID NO:1. The molecule canbe an antibody such as a monoclonal antibody or a monospecificpolyclonal antibody. In this variation, the antibody can be labeled witha detectable label.

The invention also features a composition for stimulating an immuneresponse against Sarcocystis neurona when administered to an animal. Thecomposition includes (a) an isolated agent that can specificallystimulate an immune response against a protein consisting of the aminoacid sequence of SEQ ID NO:1 when administered to an animal; and (b) apharmaceutically acceptable carrier. The composition can include anadjuvant such as an aluminum salt; an oil-in-water emulsion; acomposition including saponin; a composition including a bacterialprotein; or a cytokine.

The agent that can stimulate an immune response against Sarcocystisneurona when administered to an animal can include a nucleic acid thatcan be a first polynucleotide including a nucleotide sequence thatencodes a protein that shares at least 50% sequence identity with SEQ IDNO:1; or a second polynucleotide that binds under stringenthybridization conditions to a complement of the nucleotide sequence SEQID NO:3, wherein the second polynucleotide can be at least 30nucleotides in length. In the foregoing, the nucleic acid can be a nakedDNA, a nucleic acid incorporated into an expression vector, or apolypeptide

Unless otherwise defined, all technical terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention belongs. Commonly understood definitions ofmolecular biology terms can be found in Rieger et al., Glossary ofGenetics: Classical and Molecular, 5th edition, Springer-Verlag: NewYork, 1991; and Lewin, Genes V, Oxford University Press: New York, 1994.

By the term “gene” is meant a nucleic acid molecule that codes for aparticular protein, or in certain cases, a functional or structural RNAmolecule. For example, the SnSAG-1 gene encodes the SnSAG-1 protein.

As used herein, a “nucleic acid” or a “nucleic acid molecule” means achain of two or more nucleotides such as RNA (ribonucleic acid) and DNA(deoxyribonucleic acid). A “purified” nucleic acid molecule is one thatis substantially separated from other nucleic acid sequences in a cellor organism in which the nucleic acid naturally occurs (e.g., 30, 40,50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% free of contaminants). Theterm includes, e.g., a recombinant nucleic acid molecule incorporatedinto a vector, a plasmid, a virus, or a genome of a prokaryote oreukaryote. Examples of purified nucleic acids include cDNAs, fragmentsof genomic nucleic acids, nucleic acids produced polymerase chainreaction (PCR), nucleic acids formed by restriction enzyme treatment ofgenomic nucleic acids, recombinant nucleic acids, and chemicallysynthesized nucleic acid molecules. A “recombinant” nucleic acidmolecule is one made by an artificial combination of two otherwiseseparated segments of sequence, e.g., by chemical synthesis or by themanipulation of isolated segments of nucleic acids by geneticengineering techniques.

By the terms “SnSAG-1 gene,” “SnSAG-1 polynucleotide,” or “SnSAG-1nucleic acid” is meant a native SnSAG-1-encoding nucleic acid sequence,e.g., the native SnSAG-1 nucleic acid (SEQ ID NO:3); a nucleic acidhaving sequences from which a SnSAG-1 cDNA can be transcribed; and/orallelic variants and homologs of the foregoing. The terms encompassdouble-stranded DNA, single-stranded DNA, and RNA.

As used herein, the terms “protein” and “polypeptide” are usedsynonymously to mean any peptide-linked chain of amino acids, regardlessof length or post-translational modification, e.g., glycosylation orphosphorylation. An “purified” polypeptide is one that has beensubstantially separated or isolated away from other polypeptides in acell, organism, or mixture in which the polypeptide occurs (e.g., 30,40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 100% free of contaminants).An “isolated” polypeptide is a purified polypeptide that is not includedon a substrate (e.g., a polyacrylamide gel) with other purifiedpolypeptides from the cell or organism in which the polypeptide occurs.

By the terms “SnSAG-1 protein” or “SnSAG-1 polypeptide” are meant anexpression product of a SnSAG-1 nucleic acid (e.g., one consisting ofSEQ ID NO:3), or a protein that shares at least 50% (but preferably 55,60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99%) amino acid sequenceidentity with SEQ ID NO:3.

As used herein, “sequence identity” means the percentage of identicalsubunits at corresponding positions in two sequences when the twosequences are aligned to maximize subunit matching, i.e., taking intoaccount gaps and insertions. When a subunit position in both of the twosequences is occupied by the same monomeric subunit, e.g., if a givenposition is occupied by an alanine in each of two polypeptide molecules,then the molecules are identical at that position. For example, if 7positions in a sequence 10 amino acids in length are identical to thecorresponding positions in a second 10 amino acid sequence, then the twosequences have 70% sequence identity. Likewise, if 7 positions in asequence 10 nucleotides in length are identical to the correspondingpositions in a second 10-nucleotide sequence, then the two sequenceshave 70% sequence identity. Preferably, the length of the comparedsequences is at least 60 nucleotides, more preferably at least 75nucleotides, and most preferably 100 nucleotides. Sequence identity canbe measured using sequence analysis software (e.g., one or more of thealgorithms of MegAlign™ sequence analysis software from DNA STAR, Inc.,Madison, Wis.).

When referring to hybridization of one nucleic to another, “lowstringency conditions” means in 10% formamide, 5×Denhart's solution,6×SSPE, 0.2% SDS at 42° C., followed by washing in 1×SSPE, 0.2% SDS, at50° C.; “moderate stringency conditions” means in 50% formamide,5×Denhart's solution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in0.2× SSPE, 0.2% SDS, at 65° C.; and “high stringency conditions” meansin 50% formamide, 5× Denhart's solution, 5×SSPE, 0.2% SDS at 42° C.,followed by washing in 0.1×SSPE, and 0.1% SDS at 65° C. The phrase“stringent hybridization conditions” means low, moderate, or highstringency conditions.

A “fragment” of a SnSAG-1 nucleic acid is a portion of a SnSAG-1 nucleicacid that is less than full-length and comprises at least a minimumlength capable of hybridizing specifically with a native SnSAG-1 nucleicacid under stringent hybridization conditions. The length of such afragment is preferably at least 15 nucleotides, more preferably at least20 nucleotides, and most preferably at least 30 nucleotides of a nativeSnSAG-1 nucleic acid sequence. A “fragment” of a SnSAG-1 polypeptide isa portion of a SnSAG-1 polypeptide that is less than full-length (e.g.,a polypeptide consisting of 5, 10, 15, 20, 30, 40, 50, 75, 100 or moreamino acids of a native SnSAG-1 protein).

When referring to a nucleic acid molecule or polypeptide, the term“native” refers to a naturally-occurring (e.g., a “wild-type”) nucleicacid or polypeptide. A “homolog” of a SnSAG-1 gene is a gene sequenceencoding a SnSAG-1 polypeptide isolated from an organism other than ahuman being. Similarly, a “homolog” of a native SnSAG-1 polypeptide isan expression product of a SnSAG-1 gene homolog.

As used herein, a “SnSAG-1 marker” is any molecule whose presence in asample (e.g., a cell) indicates that a SnSAG-1 gene or protein ispresent in the sample or subject from which the sample was derived.SnSAG-1 markers include SnSAG-1 nucleic acids, SnSAG-1 proteins, andantibodies that specifically bind SnSAG-1 proteins. “Expressing aSnSAG-1 gene” or like phrases mean that a sample contains atranscription product (e.g., messenger RNA, i.e., “mRNA”) of a SnSAG-1gene or a translation product of a SnSAG-1 protein-encoding nucleic acid(e.g., a SnSAG-1 protein). A cell expresses a SnSAG-1 gene when itcontains a detectable level of a SnSAG-1 nucleic acid or a SnSAG-1protein.

By the term “antibody” is meant an immunoglobulin as well as any portionor fragment of an immunoglobulin whether made by enzymatic digestion ofintact immunoglobulin or by techniques in molecular biology. The termalso refers to a mixture containing an immunoglobulin (or portion orfragment thereof) such as an antiserum.

The term “specifically binds”, as used herein, when referring to apolypeptide (including antibodies) or receptor, refers to a bindingreaction which is determinative of the presence of the protein orpolypeptide or receptor in a heterogeneous population of proteins andother biologics. Thus, under designated conditions (e.g. immunoassayconditions in the case of an antibody), the specified ligand or antibodybinds to its particular “target” (e.g. anti-SnSAG-1 antibodyspecifically binds to an SnSAG-1polypeptide) and does not bind in asignificant amount to other proteins present in the sample or to otherproteins to which the ligand or antibody may come in contact in anorganism.

By the phrase “stimulating an immune response” is meant eliciting orincreasing the activation of a lymphocyte (e.g., a B cell or T cell) orother immune system component. The stimulation of an immune responseagainst a specific antigen can be measured as an increase in antibodytiter against that antigen or the activation of one or more lymphocyteshaving a surface receptor specific for the antigen. Activation oflymphocytes can be determined by conventional assays, e.g., theinduction of mitosis, secretion of cytokines, modulation of cell surfacemolecule expression, secretion of immunoglobulin (B cells), andincreased killing of target cells (cytotoxic T cells).

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. In the case of conflict,the present specification, including definitions will control. Inaddition, the particular embodiments discussed below are illustrativeonly and not intended to be limiting.

DETAILED DESCRIPTION

A gene encoding a S. neurona surface antigen has been cloned andsequenced. The antigen encoded by the gene has been characterized.Rabbit anti-S. neurona polyclonal antibody was used to immunoprecipitateand concentrate proteins of an isolate of S. neurona for detection ofantibodies in body fluids of clinically ill horses. The serum andcerebral spinal fluid (CSF) of diseased animals was used to identifyantigens important in natural infections. Techniques were developed toseparate parasites from host cells facilitating production a cDNAexpression library. The library was screened with both polyclonal rabbitanti-S. neurona and mass culture supernatant from hybridoma cellsproduced from mice immunized with the S. neurona isolate. A cone wasalso identified in a collection of random sequence tags prepared tocharacterize the cDNA library.

A gene fragment was cloned from the library and used as a probe toselect the full length copy of the gene encoding a major surfaceantigen, SnSAG-1, of S. neurona. The sequence data from the full lengthgene was used to generate PCR primers for producing an ampliconcontaining the open reading frame of SnSAG-1 with flanking BamH1restriction sites. This approach allowed the gene to be sub-cloned intothe BamH1 site of the expression vector pET14b which allows expressionof a His-tagged recombinant protein (i.e., His-tagged rSnSAG-1). Thisrecombinant protein migrated slightly larger on SDS-PAGE than the nativeantigen.

A monoclonal antibody that specifically binds an epitope of the 29 kDaprotein (corresponding to SnSAG-1) from cultured S. neurona merozoiteswas used to verify the presence of the epitope on the recombinantprotein. The recombinant protein was purified and used to produce amonospecific polyclonal antibody in mice and goats. The anti-SnSAG-1antisera was used to characterize the 29 kDa antigen of cultured S.neurona merozoites as a surface protein.

The cloned DNA sequence was used as a probe to determine the relativeabundance and size of the gene transcript on an RNA blot. Additionally,probing Southern blots indicated that the gene was present in genomicDNA of S. neurona but not other species (S. falcatula, T. gondii, orNeospora hughesii).

This invention encompasses compositions and methods relating to SnSAG-1.The below described preferred embodiments illustrate adaptations ofthese compositions and methods. Nonetheless, from the description ofthese embodiments, other aspects of the invention can be made and/orpracticed based on the description provided below.

Biological Methods

Methods involving conventional molecular biology techniques aredescribed herein. Such techniques are generally known in the art and aredescribed in detail in methodology treatises such as Molecular Cloning:A Laboratory Manual, 2nd ed., vol. 1-3, ed. Sambrook et al., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and CurrentProtocols in Molecular Biology, ed. Ausubel et al., Greene Publishingand Wiley-Interscience, New York, 1992 (with periodic updates). Varioustechniques using polymerase chain reaction (PCR) are described, e.g., inInnis et al., PCR Protocols: A Guide to Methods and Applications,Academic Press: San Diego, 1990: PCR-primer pairs can be derived fromknown sequences by known techniques such as using computer programsintended for that purpose (e.g., Primer, Version 0.5, ©©1991, WhiteheadInstitute for Biomedical Research, Cambridge, Mass.). The ReverseTranscriptase Polymerase Chain Reaction (RT-PCR) method used to identifyand amplify certain polynucleotide sequences within the invention wasperformed as described in Elek et al., In Vivo, 14:172-182, 2000).Methods for chemical synthesis of nucleic acids are discussed, forexample, in Beaucage and Carruthers, Tetra. Letts. 22:1859-1862, 1981,and Matteucci et al., J. Am. Chem. Soc. 103:3185, 1981. Chemicalsynthesis of nucleic acids can be performed, for example, on commercialautomated oligonucleotide synthesizers. Immunological methods (e.g.,preparation of antigen-specific antibodies, immunoprecipitation, andimmunoblotting) are described, e.g., in Current Protocols in Immunology,ed. Coligan et al., John Wiley & Sons, New York, 1991; and Methods ofImmunological Analysis, ed. Masseyeff et al., John Wiley & Sons, NewYork, 1992.

Nucleic Acids

The present invention includes the SnSAG-1 gene of S. neurona. Thenucleotide sequence of the gene encoding SnSAG-1 along with someadjacent sequences is shown herein as SEQ ID NO:2. The region of thisnucleic acid encoding the native SnSAG-1 protein (see SEQ ID NO:3) isfound in positions 73-903 (SEQ ID NO:3).

A preferred nucleic acid molecule of the invention is the native SnSAG-1polynucleotide shown herein as SEQ ID NO:3. Another nucleic acid of theinvention includes a purified nucleic acid (polynucleotide) that encodesa polypeptide having the amino acid sequence of SEQ ID NO:1. As thenative SnSAG-1 gene was cloned from a gene library of the UCD-1 isolateof S. neurona, nucleic acid molecules encoding a polypeptide of thepresent invention can be obtained from such a library or from theisolate itself by conventional cloning methods such as those describedherein.

Nucleic acid molecules of the present invention may be in the form ofRNA or in the form of DNA (e.g., cDNA, genomic DNA, and synthetic DNA).The DNA may be double-stranded or single-stranded, and ifsingle-stranded may be the coding (sense) strand or non-coding(anti-sense) strand. The coding sequence which encodes the nativeSnSAG-1 protein may be identical to the nucleotide sequence shown hereinas SEQ ID NO:3. It may also be a different coding sequence which, as aresult of the redundancy or degeneracy of the genetic code, encodes thesame polypeptide as the polynucleotide of SEQ ID NO:3. Other nucleicacid molecules within the invention are variants of the native SnSAG-1gene such as those that encode fragments, analogs and derivatives of anative SnSAG-1 protein. Such variants may be, e.g., a naturallyoccurring allelic variant of the native SnSAG-1 gene, a homolog of thenative SnSAG-1 gene, or a non-naturally occurring variant of the nativeSnSAG-1 gene. These variants have a nucleotide sequence that differsfrom the native SnSAG-1 gene in one or more bases. For example, thenucleotide sequence of such variants can feature a deletion, addition,or substitution of one or more nucleotides of the native SnSAG-1 gene.Nucleic acid insertions are preferably of about 1 to 10 contiguousnucleotides, and deletions are preferably of about 1 to 30 contiguousnucleotides.

Variant SnSAG-1 proteins displaying substantial changes in structure canbe generated by making nucleotide substitutions that cause less thanconservative changes in the encoded polypeptide. Examples of suchnucleotide substitutions are those that cause changes in (a) thestructure of the polypeptide backbone; (b) the charge or hydrophobicityof the polypeptide; or (c) the bulk of an amino acid side chain.Nucleotide substitutions generally expected to produce the greatestchanges in protein properties are those that cause non-conservativechanges in codons. Examples of codon changes that are likely to causemajor changes in protein structure are those that cause substitution of(a) a hydrophilic residue, e.g., serine or threonine, for (or by) ahydrophobic residue, e.g., leucine, isoleucine, phenylalanine, valine oralanine; (b) a cysteine or proline for (or by) any other residue; (c) aresidue having an electropositive side chain, e.g., lysine, arginine, orhistadine, for (or by) an electronegative residue, e.g., glutamine oraspartine; or (d) a residue having a bulky side chain, e.g.,phenylalanine, for (or by) one not having a side chain, e.g., glycine.

Naturally occurring allelic variants of the native SnSAG-1 gene withinthe invention are nucleic acids isolated from S. neurona that have atleast 75% (e.g., 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 97%, 98%, and 99%) sequence identitywith the native SnSAG-1 gene, and encode polypeptides having structuralsimilarity to native SnSAG-1 protein. Homologs of the native SnSAG-1gene within the invention are nucleic acids isolated from other speciesthat have at least 50% (e.g., 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 98%, and 99%) sequence identity with the native SnSAG-1gene, and encode polypeptides having structural similarity to nativeSnSAG-1 protein. Public and/or proprietary nucleic acid databases can besearched to identify other nucleic acid molecules having a high percent(e.g., 70, 80, 90% or more) sequence identity to the native SnSAG-1gene.

Non-naturally occurring SnSAG-1 gene variants are nucleic acids that donot occur in nature (e.g., are made by the hand of man), have at least50% (e.g., 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, and 99%) sequence identity with the native SnSAG-1 gene,and encode polypeptides having structural similarity to native SnSAG-1protein (e.g., those that cross react with antibodies that specificallybind the native SnMS1 protein). Examples of non-naturally occurringSnSAG-1 gene variants are those that encode a fragment of a SnSAG-1protein, those that hybridize to the native SnSAG-1 gene or a complementof to the native SnSAG-1 gene under stringent hybridization conditions,those that share at least 50% sequence identity with the native SnSAG-1gene or a complement of the native SnSAG-1 gene, and those that encodean SnSAG-1 fusion protein.

Nucleic acids encoding fragments of native SnSAG-1 protein within theinvention are those that encode, e.g., 2, 5, 10, 25, 50, 100, 150, 200,250, or more amino acid residues of the native SnSAG-1 protein. Shorteroligonucleotides (e.g., those of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 30, 50, 100, 125, 150 or 200 base pairs in length) thatencode or hybridize with nucleic acids that encode fragments of thenative SnSAG-1 protein can be used as probes, primers, or antisensemolecules. Longer polynucleotides (e.g., those of 300, 400, 500, 600,700, or 800 base pairs) that encode or hybridize with nucleic acids thatencode fragments of native SnSAG-1 protein can also be used in variousaspects of the invention. Nucleic acids encoding fragments of nativeSnSAG-1 protein can be made by enzymatic digestion (e.g., using arestriction enzyme) or chemical degradation of the full length nativeSnSAG-1 gene or variants thereof.

Nucleic acids that hybridize under stringent hybridization conditions tothe nucleic acid of SEQ ID NO:3 or the complement of SEQ ID NO:3 canalso be used in the invention. For example, such nucleic acids can bethose that hybridize to SEQ ID NO:3 or the complement of SEQ ID NO:3under low stringency conditions, moderate stringency conditions, or highstringency conditions are within the invention. Preferred suchnucleotide acids are those having a nucleotide sequence that is thecomplement of all or a portion of SEQ ID NO:3. Other variants of thenative SnSAG-1 gene within the invention are polynucleotides that shareat least 50% (e.g.,50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94,95, 96, 97, 98, and 99%) sequence identity to SEQ ID NO:3 or thecomplement of SEQ ID NO:3. Nucleic acids that hybridize under stringenthybridization conditions to or share at least 50% sequence identity withSEQ ID NO:3 or the complement of SEQ ID NO:3 can be obtained bytechniques known in the art such as by making mutations in the nativeSnSAG-1 gene, or by isolation from an organism expressing such a nucleicacid (e.g., an allelic variant).

Nucleic acid molecules encoding SnSAG-1 fusion proteins are also withinthe invention. Such nucleic acids can be made by preparing a construct(e.g., an expression vector) that expresses an SnSAG-1 fusion proteinwhen introduced into a suitable host. For example, such a construct canbe made by ligating a first polynucleotide encoding an SnSAG-1 proteinfused in frame with a second polynucleotide encoding another protein (ora HIS tag) such that expression of the construct in a suitableexpression system yields a fusion protein.

The oligonucleotides of the invention can be modified at the basemoiety, sugar moiety, or phosphate backbone, for example, to improvestability of the molecule, hybridization, etc. Oligonucleotides withinthe invention may additionally include other appended groups such aspeptides (e.g., for targeting host cell receptors in vivo), or agentsfacilitating transport across the cell membrane (see, e.g., Letsinger etal. (1989) Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre et al.(1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCT Publication No. WO88/09810, published Dec. 15, 1988), hybridization-triggered cleavageagents. (See, e.g., Krol et al. (1988) BioTechniques 6:958-976) orintercalating agents. (See, e.g., Zon (1988) Pharm. Res. 5:539-549). Tothis end, the oligonucleotides may be conjugated to another molecule,e.g., a peptide, hybridization triggered cross-linking agent, transportagent, hybridization-triggered cleavage agent, etc.

Using the nucleotide of the native SnSAG-1 gene and the amino acidsequence of the native SnSAG-1 protein previously reported, thoseskilled in the art can create nucleic acid molecules that have minorvariations in their nucleotide, by, for example, standard nucleic acidmutagenesis techniques or by chemical synthesis. Variant SnSAG-1 nucleicacid molecules can be expressed to produce variant SnSAG-1 proteins.

Polypeptides

The gene sequence shown herein as SEQ ID NO:3 encodes the native SnSAG-1protein shown herein as SEQ ID NO:1. The native SnSAG-1 protein ispredicted to have a molecular weight of 28,328 daltons and anisoelectric point (pI) of 7.48. It is a Type Ia membrane protein with aputative signal peptide, one transmembrane spanning region at its Cterminus, and is presently believed to be anchored in the merozoitemembrane via a glycophosphatidyl inositol (GPI) linkage. It has apotential cleavage site at residue 248.

The present invention provides a purified SnSAG-1 polypeptide encoded bya nucleic acid of the invention such as that shown as SEQ ID NO:3. Apreferred form of SnSAG-1 polypeptide is a purified native SnSAG-1protein that has the deduced amino acid sequence shown herein as SEQ IDNO:1. Polypeptide variants of native SnSAG-1 protein such as fragments,analogs and derivatives of the native SnSAG-1 protein are also withinthe invention. Such variants include, e.g., a polypeptide encoded by anaturally occurring allelic variant of native SnSAG-1 gene, apolypeptide encoded by an alternative splice form of the native SnSAG-1gene, a polypeptide encoded by a homolog of native SnSAG-1 gene, and apolypeptide encoded by a non-naturally occurring variant of nativeSnSAG-1 gene.

SnSAG-1 polypeptide variants have a peptide sequence that differs fromnative SnSAG-1 in one or more amino acids. The peptide sequence of suchvariants can feature a deletion, addition, or substitution of one ormore amino acids of a native SnSAG-1 protein. Amino acid insertions arepreferably of about 1 to 4 contiguous amino acids, and deletions arepreferably of about 1 to 10 contiguous amino acids.

SnSAG-1 protein fragments corresponding to one or more particular motifsand/or domains or to arbitrary sizes, for example, at least 5, 10, 25,50, 75, 100, 125, 150, 175, 200, and 250 amino acids in length arewithin the scope of the present invention. Isolated peptidyl portions ofSnSAG-1 polypeptides can be obtained by screening peptides recombinantlyproduced from the corresponding fragment of the nucleic acid encodingsuch peptides. In addition, fragments can be chemically synthesizedusing techniques known in the art such as conventional Merrifield solidphase f-Moc or t-Boc chemistry. Particularly preferred SnSAG-1 proteinfragments are those that are specifically bound by antibodies developedagainst the whole native SnMSA protein and those antibodies thatspecifically bind whole S. neurona merozoites with little or nodetectable cross-reaction to other species.

Another aspect of the present invention concerns recombinant forms ofthe SnSAG-1 polypeptides. Exemplary recombinant polypeptides of thepresent invention, in addition to native SnSAG-1 protein, are encoded bya nucleic acid that has at least 85% sequence identity (e.g., 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%) with thenucleic acid sequence of SEQ ID NO:3.

The invention also provides for reduction of SnSAG-1 polypeptides togenerate mimetics, e.g., peptide or non-peptide agents, that are able todisrupt binding of a SnSAG-1 protein to other proteins or molecules withwhich the native SnSAG-1 protein interacts. For instance,non-hydrolyzable peptide analogs of such residues can be generated usingbenzodiazepine (e.g., see Freidinger et al. in Peptides: Chemistry andBiology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands,1988), azepine (e.g., see Huffman et al. in Peptides: Chemistry andBiology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands,1988), substituted gamma lactam rings (Garvey et al. in Peptides:Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden,Netherlands, 1988), keto-methylene pseudopeptides (Ewenson et al. (1986)J. Med. Chem. 29:295; and Ewenson et al. in Peptides: Structure andFunction (Proceedings of the 9th American Peptide Symposium) PierceChemical Co. Rockland, Ill., 1985), beta-turn dipeptide cores (Nagai etal. (1985) Tetrahedron Lett 26:647; and Sato et al. (1986) J. Chem. Soc.Perkin. Trans. 1:1231), and b-aminoalcohols (Gordon et al. (1985)Biochem. Biophys. Res. Commun. 126:419; and Dann et al. (1986) Biochem.Biophys. Res. Commun. 134:71). SnSAG-1 polypeptides may also bechemically modified to create SnSAG-1 derivatives by forming covalent oraggregate conjugates with other chemical moieties, such as glycosylgroups, lipids, phosphate, acetyl groups and the like. Covalentderivatives of SnSAG-1 can be prepared by linking the chemical moietiesto functional groups on amino acid side chains of the protein or at theN-terminus or at the C-terminus of the polypeptide.

The present invention further pertains to methods of producing thesubject SnSAG-1 polypeptides. For example, a host cell (e.g., E. coli)transfected with a nucleic acid vector directing expression of anucleotide sequence encoding the subject polypeptides can be culturedunder appropriate conditions to allow expression of the peptide tooccur. The cells may be harvested, lysed, and the protein isolated. Arecombinant SnSAG-1 polypeptide can be isolated from host cells usingtechniques known in the art for purifying proteins includingion-exchange chromatography, gel filtration chromatography,ultrafiltration, electrophoresis, and immunoaffinity purification withantibodies specific for such protein.

After SnSAG-1 protein has been expressed in a cell, it can be isolatedusing any immunoaffinity chromatography. For instance, an anti-SnSAG-1antibody (e.g., produced as described below) can be immobilized on acolumn chromatography matrix, and the matrix can be used forimmuno-affinity chromatography to purify SnSAG-1 protein from celllysates by standard methods (see, e.g., Ausubel et al., supra). Afterimmuno-affinity chromatography, SnSAG-1 protein can be further purifiedby other standard techniques, e.g., high performance liquidchromatography (see, e.g., Fisher, Laboratory Techniques In BiochemistryAnd Molecular Biology, Work and Burdon, eds., Elsevier, 1980). Inanother embodiment, SnSAG-1 protein is expressed as a fusion proteincontaining an affinity tag (e.g., GST) that facilitates itspurification.

Antibodies

SnSAG-1 polypeptides (or immunogenic fragments or analogs thereof) canbe used to raise antibodies useful in the invention. Such polypeptidescan be produced by recombinant techniques or synthesized as describedabove. In general, SnSAG-1 polypeptides can be coupled to a carrierprotein, such as KLH, as described in Ausubel et al., supra, mixed withan adjuvant, and injected into a host mammal. Antibodies produced inthat animal can then be purified by peptide antigen affinitychromatography. In particular, various host animals can be immunized byinjection with a SnSAG-1 polypeptide or an antigenic fragment thereof.Commonly employed host animals include rabbits, mice, guinea pigs, andrats. Horses are preferred for the production of equine antibodies andfor many of the methods described herein. Various adjuvants that can beused to increase the immunological response depend on the host speciesand include Freund's adjuvant (complete and incomplete), mineral gelssuch as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanin, and dinitrophenol. Other potentially usefuladjuvants include BCG (bacille Calmette-Guerin) and Corynebacteriumparvum.

Polyclonal antibodies are heterogeneous populations of antibodymolecules that are contained in the sera of the immunized animals.Antibodies within the invention therefore include polyclonal antibodiesand, in addition, monoclonal antibodies, single chain antibodies, Fabfragments, F(ab′)₂ fragments, and molecules produced using a Fabexpression library. Monoclonal antibodies, which are homogeneouspopulations of antibodies to a particular antigen, can be prepared usingthe SnSAG-1 polypeptides described above and standard hybridomatechnology (see, for example, Kohler et al., Nature 256:495, 1975;Kohler et al., Eur. J. Immunol. 6:511, 1976; Kohler et al., Eur. J.Immunol. 6:292, 1976; Hammerling et al., “Monoclonal Antibodies and TCell Hybridomas,” Elsevier, N.Y., 1981; Ausubel et al., supra). Inparticular, monoclonal antibodies can be obtained by any technique thatprovides for the production of antibody molecules by continuous celllines in culture such as described in Kohler et al., Nature 256:495,1975, and U.S. Pat. No. 4,376,110; the human B-cell hybridoma technique(Kosbor et al., Immunology Today 4:72, 1983; Cole et al., Proc. Natl.Acad. Sci. USA 80:2026, 1983), and the EBV-hybridoma technique (Cole etal., “Monoclonal Antibodies and Cancer Therapy,” Alan R. Liss, Inc., pp.77-96, 1983). Such antibodies can be of any immunoglobulin classincluding IgG, IgM, IgE, IgA, IgD and any subclass thereof. A hybridomaproducing a mAb of the invention may be cultivated in vitro or in vivo.The ability to produce high titers of mAbs in vivo makes this aparticularly useful method of production.

Techniques described for the production of single chain antibodies(e.g., U.S. Pat. Nos. 4,946,778, 4,946,778, and 4,704,692) can beadapted to produce single chain antibodies against an SnSAG-1polypeptide, or a fragment thereof. Single chain antibodies are formedby linking the heavy and light chain fragments of the Fv region via anamino acid bridge, resulting in a single chain polypeptide.

Antibody fragments that recognize and bind to specific epitopes can begenerated by known techniques. For example, such fragments include butare not limited to F(ab′)₂ fragments that can be produced by pepsindigestion of the antibody molecule, and Fab fragments that can begenerated by reducing the disulfide bridges of F(ab′)₂ fragments.Alternatively, Fab expression libraries can be constructed (Huse et al.,Science 246:1275, 1989) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

Preferably, SnSAG-1 polypeptide selective antibodies of the inventionare produced using fragments of SnSAG-1 that lie outside highlyconserved regions and appear likely to be antigenic, by criteria such ashigh frequency of charged residues. Cross-reactive anti-SnSAG-1antibodies are produced using a fragment of SnSAG-1 that is conservedamongst members of this family of proteins. In one specific example,such fragments are generated by standard techniques of PCR, and are thencloned into the pGEX expression vector (Ausubel et al., supra). Fusionproteins are expressed in E. coli and purified using a glutathioneagarose affinity matrix as described in Ausubel, et al., supra.

Once produced, polyclonal or monoclonal antibodies or fragments thereofcan be tested for specific SnSAG-1 recognition by Western blot orimmunoprecipitation analysis by standard methods, for example, asdescribed in Ausubel et al., supra. Antibodies that specificallyrecognize and bind to SnSAG-1 polypeptide are useful in the invention.

The antibodies of the invention can be used, for example, in thedetection of an SnSAG-1 polypeptide in a biological sample (e.g., serumor CSF from a horse). Antibodies also can be used in a screening assayto measure the effect of a candidate compound on expression orlocalization of SnSAG-1 polypeptides. Additionally, such antibodies canbe used to interfere with the interaction of SnSAG-1 polypeptides andother molecules that bind SnSAG-1 polypeptides.

Methods of Detecting S. neurona

This invention is also related to the use of a SnSAG-1 nucleic acid aspart of a diagnostic assay for detecting the presence of S. neurona in abiological sample (e.g., an S. neurona infection is a horse). Forexample, nucleic acid is isolated from a biological sample such as fromthe CSF, blood, urine, saliva, tissue biopsy or necropsy material takenfrom an animal. The isolated nucleic acid may be used directly fordetection or may be amplified enzymatically by using PCR (Saiki et al.,Nature, 324: 163-166 (1986)) prior to analysis. RNA or cDNA may also beused for the same purpose. As an example, oligonucleotides complementaryto the nucleic acid encoding an SnSAG-1 polypeptide can be used asprobes or PCR primers to identify and analyze the isolated nucleic acidfor the presence of nucleotide sequences encoding the SnSAG-1polypeptide.

As one example, nucleic acid from a biological sample (e.g., nervetissue or CSF from a horse) is isolated from the sample (e.g., by PCRamplification) and then immobilized on a substrate (e.g., by UVcross-linking to a blotting membrane). The substrate is then contactedwith a labeled (e.g., chemiluminescent or radioactive) nucleic acidprobe that hybridizes to an SnSAG-1 nucleic acid under stringenthybridization conditions. After washing the substrate to remove unboundprobe, the amount of labeled probe bound to the substrate is assessed(e.g., by exposing the substrate to photographic or X-ray film). Bindingof probe to the immobilized nucleic acid indicates the presence of anucleic acid encoding an SnSAG-1 polypeptide in the biological sample.Specific nucleic acid sequences may also be detected using methods suchas other types of hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,Restriction Fragment Length Polymorphism analysis (RFLP)), Northernblotting of mRNA, and Southern blotting of genomic DNA). Assays used todetect the presence or amount of SnSAG-1 polypeptide in a biologicalsample are also within the invention. Examples of such assays includeradioimmunoassay, competitive-binding assays, Western Blot analysis,ELISA assays and “sandwich” assays. As one example, a typical ELISAassay can be performed by first preparing an antibody that specificallybinds to an SnSAG-1 polypeptide. The antibody, which can be monoclonalor polyclonal, is then conjugated to a detectable label such as anenzyme (e.g., horseradish peroxidase). A biological sample to be tested(e.g., CSF from a horse) is then immobilized on a solid substrate, e.g.,by incubating the sample on a polystyrene dish that has been treated tobind proteins contained in the sample. Free protein binding sites on thesubstrate are then blocked by adding a non-specific protein like albuminon the substrate. After washing the substrate, the anti-SnSAG-1 antibodyis contacted to the substrate under conditions that allow the antibodyto bind any SnSAG-1 polypeptide attached to the substrate. The substrateis then washed to remove any unbound antibody. Antibody remainingattached to the substrate is then detected to provide a quantitativeestimate of the amount of SnSAG-1 polypeptide in the biological sample.Attached antibody can be detected via the detectable label. For example,horseradish peroxidase labeled antibodies can be detected by adding anappropriate substrate that produces a color change in the presence ofhorseradish peroxidase. As another example, a competition assay may beemployed wherein antibodies that specifically bind a SnSAG-1 polypeptideare attached to a solid support and labeled-SnSAG-1 protein and a samplederived from a biological sample are passed over the solid support. Theamount of label not binding to the solid support can be detected, forexample by liquid scintillation chromatography, and correlated to aquantity of SnSAG-1 polypeptide in the sample (more label not bound tothe support correlates with more SnSAG-1 polypeptide in the sample).

Methods of Detecting S. neurona-specific Antibodies

Methods of detecting the presence of antibodies that specifically bindto a SnSAG-1 polypeptide are also within the invention. As one exemplarymethod, an SnSAG-1 polypeptide (e.g., rSnMSA protein or fragmentthereof) is immobilized on a substrate (e.g., immobilized on a blottingmembrane or in the wells of microtiter plate). After washing, abiological sample (e.g., blood or serum from a horse) is added to thesubstrate under conditions that allow any anti-SnSAG-1 antibodiespresent in the sample to bind to the immobilized SnSAG-1 polypeptide.After again washing the substrate to remove any unbound antibody, areagent that detects bound antibody is added to the substrate andallowed to bind any antibody attached to the substrate. The reagent can,for example, take the form of a labeled-secondary antibody that binds toanti-SnSAG-1 antibody (e.g., for horse blood, the antibody could be ahorseradish peroxidase-conjugated anti-horse immunoglobulin antibody).Unbound reagent is then washed away, and the amount of attached reagentis assessed as an indication of the presence of anti-SnSAG-1 antibodiesin the sample (more reagent correlates with more antibody). Variationsof the foregoing and other methods of detecting anti-SnSAG-1 antibodieswithin the invention include radioimmunoassay, competitive-bindingassays, Western Blot analysis, ELISA assays and “sandwich” assays.

Kits for Detecting S. neurona

The invention also provides a kit for detecting S. neurona eitherdirectly or indirectly (e.g., by detecting antibodies produced againstS. neurona by an animal's immune system). The kit can feature one ormore of the nucleic acids, polypeptides, or antibodies of the invention;various devices and reagents for performing one of the assays describedherein (e.g., blotting membranes, microtiter plates, labeled-secondaryantibodies, etc.); and/or printed instructions for using the kit todetect S. neurona or an antigen or nucleic acid thereof.

Vaccines-method of Inducing an Immune Response

The invention also includes a composition for use in a vaccine againstS. neurona. In one embodiment, the composition includes an SnSAG-1polypeptide (e.g., native SnSAG-1 protein) and an adjuvant such asFreund's adjuvant (complete and incomplete), aluminum hydroxide, asurface active substance (e.g., lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol). Such a composition can be injected into an animal toinduce an immune response (e.g., production of antibodies against theSnSAG-1 polypeptide or a cytotoxic T cell response). Nucleic acidswithin the invention (e.g., that of SEQ ID NO:3) can also be used as avaccine by adapting known techniques. See, e.g., Wolff et al., Science247, 1465-1468, 1990.

Vaccines within the invention include an antigenic agent which can takethe form of any substance that can evoke or increase an immune responseagainst SnSAG-1 when introduced into a subject. Typical immune responsesinclude (a) the production of, or increase in titer of, antibodies thatspecifically bind SnSAG-1 and (b) the activation of T lymphocytes (e.g.,to kill a target cell or provide help in the activation of antibodyproduction in B lymphocytes). A number of different antigenic agentshave been shown to be effective in stimulating an immune responseagainst a protein antigen, including, for example, protein-andpeptide-based vaccines, tumor-cell vaccines, dendritic cell/gene therapyvaccines and DNA/viral vaccines. See, e.g., Greten, T. F. and E. M.Jaffee, J. Clin. Oncol., 17: 1047-1060, 1999. In addition to theforegoing, various substances such as adjuvants and excipients/carrierscan be included in the vaccine compositions of the invention tonon-specifically enhance the antigen-specific immune response stimulatedby the antigenic agent and to facilitate delivery of the othercomponents of the vaccine to a subject.

Protein/Peptide Based Vaccines

The antigenic agent for use in the vaccines of the invention can takethe form of the native SnSAG-1 protein (SEQ ID NO:1) or a peptidefragment thereof. Vaccines made with the whole protein antigen areadvantageous because they have the capability of stimulating an immuneresponse against all of the potential antigenic sites expressed by theprotein. Vaccines made with peptide antigens (e.g., 7-15 or 8-12contiguous amino acids of the whole protein), on the other hand, willgenerally stimulate an immune response against fewer than all of thepotential antigenic sites expressed by the protein. Peptide-basedvaccines are sometimes advantageous over whole protein-based vaccineswhere it is desired to more specifically target the stimulated immuneresponse, e.g., to avoid undesired cross reactions. For example,peptides for use in the vaccine can be selected to correspond to (1)specific epitopes of the antigens that are known to be presented by MHCclass I or MHC class II molecules, or (2) a modified form of an epitopethat either exhibits an increased stability in vivo or a higher bindingaffinity for an MHC molecule than the native epitope, while still beingcapable of specific activation of T-cells. See, Ayyoub et al., J. Biol.Chem., 274: 10227-10234, 1999; Parkhurst et al., Immunol., 157:2539-2548, 1996. Peptide-based vaccines have been shown to circumventimmune tolerance to the intact proteins. Disis et al., J. Immunol., 156:3151-3158, 1996. In addition to vaccines composed of only one type ofpeptide fragment, other vaccines within the invention also include thosemade up of a cocktail of several different peptides derived from thenative SnSAG-1 protein.

Vaccines within the invention can include a SnSAG-1 protein other thanthe native SnSAG-1 protein as an antigenic agent. For example, variantsof the native SnSAG-1 protein such as fragments, analogs and derivativesof native SnSAG-1 are also contemplated for use as an antigenic agent inthe vaccines of the invention. Such variants include, e.g., apolypeptide encoded by a naturally occurring allelic variant of thenative SnSAG-1 gene, a polypeptide encoded by a homolog of the nativeSnSAG-1 gene, and a polypeptide encoded by a non-naturally occurringvariant of the native SnSAG-1 gene. Preferred versions of such variantsare those that are able to stimulate a protective immune response tonative SnSAG-1 upon administration to a subject as part of a vaccine.

Nucleic Acid-based Vaccines

Nucleic acid-based vaccines are known to elicit a prominentcell-mediated immune response. See, e.g., Donnely et al., 1997;Rosenberg, S. A., Immunity 10:281, 1999. Thus, in addition toprotein/peptide based vaccines, the antigenic agent for use in thevaccines of the invention can take the form of a nucleic acid that canstimulate an immune response against SnSAG-1 when administered to asubject. Examples of such nucleic acids include those that encode thenative SnSAG-1 protein such as the nucleic acid shown herein as SEQ IDNO:3, a variant of the native SnSAG-1, or a peptide fragment of thatnative or variant SnSAG-1. Vaccines made with a nucleic acid thatencodes the whole protein antigen are advantageous because they have thepotential for stimulating an immune response against all of thedifferent antigenic sites expressed by the protein. Vaccines made with anucleic acid that encodes a peptide antigen (e.g., 7-15 amino acids ofthe whole protein), on the other hand, will generally stimulate animmune response against fewer than all of the potential antigenic sitesexpressed by the protein.

The form of the nucleic acid used in a vaccine of the invention can beany suitable for stimulating an immune response SnSAG-1 whenadministered to a subject. For example, the nucleic acid can be in theform of “naked DNA” or it can be incorporated in an expression vector.

Nucleic acids that are most immunogenic in a subject can be determinedby preparing several of a particular SnSAG-1 nucleic acid (e.g., onethat encodes the whole antigen or peptide fragments thereof),administering the subject (or a series of genetically similar suchsubjects) such nucleic acids in a vaccine composition (e.g., as nakednucleic acid or in an expression vector in a suitable carrier), andanalyzing the subject(s) for the stimulation of an immune response.Those nucleic acids that induce the desired response can then beselected.

Naked Nucleic Acid Vaccines

The invention provides for the use of naked nucleic acid vaccines tostimulate an immune response against SnSAG-1. Representative nakednucleic acid vaccines for use in this method include a DNA encoding oneor more immunogenic portions of SnSAG-1 along with sufficient other 5′and 3′ elements to direct expression of the foregoing. The use of nakednucleic acids for stimulating both class I and class II restrictedimmune responses against a particular protein is known in the art. See,e.g., Rosenberg, S. A., Immunity 10:281, 1999; Ulmer et al., Science,259:1745, 1993; Donnelly et al., Ann. NY Acad. Sci., 772:40, 1995;Scheurs et al., Cancer res. 58:2509, 1998; Hurpin et al., Vaccine16:208, 1998; Lekutis et al., J. Immunol. 158:4471, 1997; Manickan etal., J. Leukoc. Biol. 61:125, 1997. These methods can be adapted for usein the present invention by using a nucleic acid encoding one or moreimmunogenic portions of SnSAG-1. Naked nucleic acid vaccines can beadministered to a subject by any suitable technique. For example, nakedDNA encoding a peptide portion of SnSAG-1 can be injected into musclecells of a subject or naked DNA-coated gold particles can be introducedinto skin cells (to be taken up by dendritic cells) of a subject using agene gun.

Expression Vector Vaccines

The invention also provides for the use of expression vector vaccines tostimulate an immune response against SnSAG-1. In a typical applicationof this technique, a nucleic acid encoding one or more peptide orprotein antigens of SnSAG-1 is incorporated into a vector that allowsexpression of the antigen(s) in a host cell (e.g., a cell inside asubject or administered to a subject). The nucleic acid encoding theantigen(s) is generally be under the operational control of othersequences contained within the vector such as a promoter sequences(e.g., tissue specific, constitutively active, or inducible) or enhancersequences. The antigen(s) encoded by the vector are expressed when thevector is introduced into a host cell in a subject. After expression,the antigen(s) can associate with an MHC molecule for presentation toimmune system cells such as T lymphocytes, thus stimulating an immuneresponse. See, e.g., Corr et al., J. Exp. Med. 184:1555 (1996). Vectorsfor use in the invention can be any capable of expressing an encodedantigen(s) in a subject. For example, vectors derived from bacterialplasmids and viruses may be used. Representative viral vectors includeretroviral, adenoviral, and adeno-associated viral vectors.

Administering Vaccines to a Subject

The vaccine compositions of the present invention can be used in amethod for stimulating an immune response against SnSAG-1 in a subject(e.g., a horse). In this method, an vaccine compositon of the inventioncan be administered to a subject by any method that stimulates theaforesaid immune response. The exact method selected is determined bythe particular vaccine composition to administered. For parenteraladministration by injection, the injection can be in situ (i.e., to aparticular tissue or location on a tissue, e.g., into a tumor or lymphnode), intramuscular, intravenous, intraperitoneal, or by anotherparenteral route. For example, for a protein/peptide based vaccine thevaccine may be administered by subcutaneous or intradermal injection. Insome cases other routes can be used, e.g. intravenous injection,intraperitoneal injection, or in situ injection into target tissue.

Naked nucleic acid vaccines or expression vector vaccines may beadministered by intramuscular injection. Cell-based vaccines can beintroduced into an animal by any suitable method, e.g., subcutaneousinjection. In addition to parenteral routes, the vaccines of theinvention can also be administered by a non-parenteral route, e.g, byoral, buccal, urethral, vaginal, or rectal administration.

Formulations for injection may be presented in unit dosage form, forexample, in ampoules or in multi-dose containers, with an addedpreservative. The compositions may take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the vaccine compositions may be in powder form(e.g., lyophilized) for constitution with a suitable vehicle, forexample, sterile pyrogen-free water, before use.

To facilitate delivery of the antigenic compositions (e.g., antigenicagent plus adjuvant) of the invention to an animal, the antigeniccompositions can be mixed with a pharmaceutically acceptable carrier orexcipient. Examples of such pharmaceutically acceptable carriers andexcipients include diluents such as water, saline, citrate bufferedsaline, phosphate buffered saline, acetate buffered saline, andbicarbonate buffered saline; and stabilizing agents such as amino acids,alcohols, proteins (for example, serum albumin), EDTA, mannitol,sorbitol, and glycerol. To minimize the chance of infection or adversereaction when administered to a subject, carriers and excipients arepreferably sterile and pyrogen-free. USP grade carriers and excipientsare particularly preferred for delivery of vaccine compositions to humansubjects. The vaccine compositions can also be formulated for long-termrelease as a depot preparation by adding the antigenic agent to suitablepolymeric or hydrophobic materials or ion exchange resins. They can alsobe made by preparing the vaccine composition as a sparingly solublederivative. Depot preparations can be administered to a subject byimplantation (e.g., subcutaneous or intramuscular surgical implantation)or by injection. Methods for making the foregoing formulations are wellknown and can be found in, for example, Remington's PharmaceuticalSciences.

Dosing

The vaccine compositions of the invention are preferably administered toa subject in an amount sufficient to stimulate an immune responseagainst S. neurona in the subject, and not cause an overly toxic effect.Such a therapeutically effective amount can be determined as describedbelow.

Toxicity and therapeutic efficacy of the vaccines utilized in theinvention can be determined by standard pharmaceutical procedures, usingeither cells in culture or experimental animals to determine the LD₅₀(the dose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio LD₅₀/ED₅₀. Vaccines that exhibit largetherapeutic indices are preferred. While those that exhibit toxic sideeffects may be used, care should be taken to design a delivery systemthat minimizes the potential damage of such side effects. The dosage ofpreferred vaccines lies preferably within a range that include an ED₅₀with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized.

The vaccines of the invention can be administered to a subject usingvarious different vaccination schedules. For example, a nucleic acidvaccine might be administered to a subject only once, while aprotein/peptide-based vaccine might be administered to the subject onmultiple occasions (1, 2, 3, 4, 5 or more times). For example, in aneffort to stimulate a strong immune response, a first dose of a vaccinecompositions of the invention may be administered to a subject at least24 hours before a second (booster) dose is administered to the subject.

Vaccine Kits

The invention also provides kits for stimulating an immune responseagainst S. neurona in a subject. Such kits can include a containerholding one or more of the antigenic agents described above in apharmaceutically acceptable form. The antigenic agent(s) in thecontainer can be in liquid form (e.g., as a solution) or in solid form(e.g., as a lyophilized or desiccated powder). Where, for example, theantigenic agent is a solid, the kits within the invention can furtherinclude a container holding a pharmaceutically acceptable solution(e.g., sterile saline with or without dextrose) for reconstituting thesolid into a liquid suitable for injection. The kits of the inventioncan further include (a) one or more devices to administer the antigenicagent, e.g., a needle or syringe, a packaged alcohol pad, etc.; and/or(b) printed instructions for using the kit.

EXAMPLES

The present invention is further illustrated by the following specificexamples. The examples are provided for illustration only and are not tobe construed as limiting the scope or content of the invention in anyway.

Example 1 Preparation of Sporocysts for In Vitro Culture

Sporocysts of S. neurona were obtained by scraping the mucosa of feralFlorida opossums that had been killed on the roadways. Mucosal scrapingswere stored in antibiotic media at 4° C. until used. Murphy A. J. andMansfield, L. S., 1999 Journal of Parasitology 85(5):979-981.Sarcocystis neurona sporocyst isolates were selected from among thoseidentified using DNA marker analysis. Dame J. B. et al. 1995 Journal ofParasitology 81(6):930-935. Prior to being placed in culture, sporocystswere treated in 5% sodium hypochlorite (bleach) for 5 min and washed intap water by repeated cycles of centrifugation (300×g, 10 min) andresuspension until the smell of bleach was undetectable. Sporocysts werefloated on an 20/30/60% isosmotic colloidal silica step gradient(Percoll®). Sporocysts obtained at the 30/60 interface were washed twicein PBS, as described above, and stored short term at 4° C. Sporozoiteswere excysted by either of two methods: 1) ˜100 sporocysts wereresuspended in 100 μl horse bile containing 2 μl trypsin (5 units/ml)and incubated for 4-6 hours at 37° C. under a 5% CO₂ 95% air atmosphereSporozoites and unexcysted sporocysts were collected by centrifugationas above, washed once in phosphate buffered saline, pH 7.2 (PBS) andresuspended in a final volume of 100 μl PBS. 2) ˜100 sporocysts wereresuspended in PBS containing proteinase K (1 mg/ml) and 1% SDS andincubated for 10 min. at 37° C. Sporocysts were pelleted bycentrifugation at 300×g for 10 min. and washed by resuspension in PBSwithout additives 3 times. Finally, the pellet was resuspended in 200 μldimethyl sulfoxide (DMSO) freeze media (Fisher) and incubated for twohours to overnight at −20° C.

Half of the excysted sporocyst preparation were added to a 25 cm² flaskwith a freshly trypsinized, 60% confluent monolayer of BM cells inDulbecco's medium containing 10% horse serum, 100 units/ml penicillin,100 units/ml streptomycin, 1 mM pyruvate and 1 mM glutamate. The culturewas maintained at 38° C. in an atmosphere of 5% CO₂, 95% air. Theculture medium was changed at 24 hours and then at 2-4 day intervals.Parasite development was monitored by direct microscopic observationwith a Nikon inverted microscope at 1-3 day intervals.

Example 2 Ionophore A23187—Stimulated Merozoite Release and Purification

A stock solution of calcium ionophore A23187 (Sigma) was prepared indimethylsulfoxide to a final concentration of 1 mg/ml and stored at −20°C. Infected cell monolayers (see example 1) at 12 days post infectionwere washed three times with PBS or Hanks Balanced Salts Solution(HBSS), and 10 ml of A23187 (1 μM in HBSS) was added to the washedmonolayer and incubated at 37° C. for 40 min. in 5% CO₂, 95% air. Freemerozoites were collected by centrifugation and washed in PBS as above.Parasites released by this method were examined by density gradientcentrifugation, but further separation from host cell debris by densitygradient centrifugation was not routinely necessary. For furtherpurification, parasites recovered from the supernatant were isolatedfrom host cell debris on a discontinuous buoyant density gradient usingIodixanol (Optiprep) in PBS or HBSS. Merozoites were suspended gently in1.0 ml PBS and were layered onto a preformed, three step discontinuousgradient with layers of 1.03, 1.04 and 1.06 g/ml in a 15 ml round bottomcentrifuge tube. The gradient was centrifuged at 1000×g for 25 min at20° C. with the brake off. Fractions containing particulate material ateach interface were collected and examined microscopically. Thefractions(s) containing significant amounts of merozoites were collectedfor use.

Collection of parasites free in the culture medium was enhanced 90 foldby incubation of infected host cells for 40 min in 1 μM A23187 prior tocollecting the culture supernatant. Parasitized host cells that releasedmerozoites in response to ionophore treatment were BM, BM0617, HL, BT,and ED cells. Infected GT, HFF, CHO, BHK, and primary EM cells wererefractory for the release of parasites under the same conditions. Therelease of parasites in response to ionophore treatment was optimal at10 to 12 days post infection, just after a few parasites were firstobserved free in the culture supernatant. Although difficult toaccurately determine the percentage of individual merozoites released bythis treatment, it was a large proportion. No mature schizonts werevisible in a microscopic examination of treated cultures. Selectivedisruption of the parasitized-host-cell membrane was seen in electronmicrographs. Initially, the host cell increased in size, becamevacuolated, and had small breaks in the membrane. As the plasma membranebecame more permeable, empty membrane bound vacuoles or vacuoles withperipheral ribosomes were released into the media. The host cell becamelong and cytoplasmic volume decreased. Parasites were observed to movebeginning at 10 minutes and continuing until their release at fortyminutes. The merozoites undulated hyperactively in this media, but withthe removal of the ionophore by addition of culture media, releasedparasites and the host cells recovered a normal appearance and activity.Ionophore-treated parasites remained animated and readily infected newcells when incubated onto a fresh monolayer. The difference noted inelectron micrographs of ionophore treated parasites was that theyappeared to have more prominent micronemes than untreated parasites. Theseparation of parasites proteins after they are treated with ionophorewas observed, however coomassie blue staining is not sufficient todistinguish parasite antigens from those of the host cells.

Parasites released from the host cell monolayer by A23187 were single,hyperactive, and entered cells readily. When A23187-treated parasiteswere used as the inoculum, the infected host cells harbored an abundanceof mature schizonts in 3 to 5 days. The extracellular parasitesremaining after washing off the ionophore did not re-invadeionophore-treated host cells, but increased in size while the fewmerozoites that remained in host cells formed mature schizonts in fivedays continuing the infection. Ten days after ionophore treatment, theparasites in the monolayer were unresponsive to calcium ionophore,whereas at thirty days, ionophore treatment again elicited parasiterelease. During this second ionophore treatment, many host cells werereleased into the supernatant.

Example 3 S. neurona Growth Rate and Response to Ionophore Treatment

Sarcocystis neurona was grown in 11 cell lines to determine growth rateand response to ionophore treatment of the monolayer. Sarcocystisneurona merozoites replicated in two different bovine monocyte lines[BM, laboratory stock culture and BM 0617 (American Type CultureCollection, Rockville, Md., USA) CRL 0617], bovine turbinate cells (BTcells, ATTC CRL 1390), human lung cells (HL cells, ATTC CCL 201-8Lu),human foreskin fibroblasts (HFF, ATTC CRL 2450), Chinese hamster ovarycells (CHO cells, ATTC CCL 61), bovine kidney (MDBK cells, ATTC CCL 22),goat tumor cells (GT cells, a gift of Dr. Jack Gaskin), equine dermalcells (ED cells, ATTC CRL 6288), and equine monocytes (EM, primaryculture from peripheral blood). Parasite growth in each of these hostcell lines was observed over a 30 day period starting from an inoculumof 2000 parasites collected from the culture supernatant of BM cells.

Typically exuberant growth with rosette formation at five days postinfection when sub-cultured from BM 0617 cells. Formation of rosetteswas first observed 3 days post infection with release and re-invasion ofnew cells occurring at five days post infection. The efficiency ofinfection was increased when freshly trypsinized host cells were placedin sufficient numbers in the culture flask to establish a 60% confluentmonolayer immediately before merozoites were added. This increase innumbers of merozoites entering host cells improved the yield ofparasites and shortened by two weeks the length of time required forculture prior to harvesting the merozoites. Infection of BT cells bymerozoites was increased by 50% using scraped cells from thirty day postinfection as inoculum rather that the supernatant from the samecultures.

Example 4 Examination of Parasites Found Free in Culture vs. Inside HostCells

Studies of the replication of the parasite were performed as follows.Approximately 220 S. neurona merozoites recovered from a culturesupernatant were added to 5000 Human Lung (HL) cells seeded and growingon Thermonax coverslips in 24 well plates to evaluate the growth andnatural release of parasites. Every three days, the 2 ml supernate wasremoved and evaluated by cytospin and the number of merozoites presentcounted. The corresponding coverslip was fixed in methanol and stainedwith Giemsa and the total number of intracellular parasites and thoseextracellular, but associated with the cover slip, were counted.

Cultures of parasites replicating in HL cells were examined to followthe number of parasites free in the culture supernatant as compared withthose found inside host cells during normal parasite growth. Themajority of the parasites were released at 21 days in this experiment.

Lung cells at 60% confluence were seeded at a density of 0.1 merozoitesper host cell and parasite growth was monitored microscopically for 30days. Free parasites were not observed until 10 days post infection.During most of the period of observation, the percentage ofextracellular parasites was less than 10%. On day 28, the largest numberof free parasites was counted; but the next day the numbers ofextracellular parasites was back to less than 10%. Duplicate 75 cmflasks were grown for 12 days in HL cells and treated with HBSS or HBSSwith 1 μM A23187. Free parasites were collected by centrifugation andcounted with a hemocytometer.

Example 5 Molecular Analysis of Parasite Cultures

DNA analysis by RAPD markers 33/54 separated the samples into thosesimilar to S. neurona, but different than S. falcatula, and those thatwere falcatula-like or 1085-like as described by Tanhauser S., et al.1999 Journal of Parasitology 85(2):221-228. Washed merozoites werepelleted by centrifugation for five minutes at 16,000 g in amicrocentrifuge. The pellet was resuspended in 100 μl of lysis buffer(50 mM KCL, 10 mM Tris-HCL, pH 9.0, 1.5 mM MgCl₂, 1% Triton X-100, 1%Tween-20, 1 μM Proteinase K), incubated two hours at 56° C., and thenboiled for two min. to inactivate the Proteinase K. The tube wascentrifuged to remove particulate matter and the supernate was useddirectly as template in a polymerase chain reaction (PCR). A PCRamplification consisting of the following reagents assembled on ice in athin-walled microfuge tube: 3 μl DNA template, 5 μl 10×PCR buffer, 3 μldNTP mix (2.5 mM each), nuclease free water to 47 μl, 2 μl 33/54 RAPDscreening primers. The PCR reaction was 94° C. for three min. with hotstart, 35 cycles: 94° C. 30 sec, 45° C. 1 min., 72° C. 45 sec, followedby 6 min. at 72° C. The resulting PCR reaction was analyzed on a 1%agarose gel in 1×TBE with ethidium bromide (0.5 μg/ml) added to therunning buffer. These RAPD markers produced a PCR product 1,100 bp.Restriction endonuclease digestions with DraI resulted in a fragment of884 and 216 bp for S. neurona, and digestions with Hinf I resulted infragments 745 and 355 for S. falcatula.

Example 6 2D Electrophoresis and Immunoprecipitation of S. neuronaAntigens

Sarcocystis neurona was grown and washed as described in Ellison, S. P.,et al. 2001 International Journal of Parasitology, followed by one washin carbonate buffer (50 mM Na₂CO₃, 0.85% NaCl, pH 7.4) and centrifugedat 300×g for 10 min. Merozoites were then washed twice in Na₂CO₃ bufferat 4° C. and placed in lysis buffer (50 mM Tris-HCl, pH 7.6, 1%TritonX-100, 1 mM EDTA) at 4° C. for 30 min. Parasite lysate wascentrifuged at 3000×g for 30 min. at 4° C. The pellet was resuspended in2DE (two dimensional electrophoresis) sample buffer (8M urea, 100 mMdithiothreitol, 4% CHAPS, 0.5% carrier amphylolytes pH 3-10, and 40 mMTris-HCl). The lysate volume was brought to 1 ml with dilution buffer towhich 10 μl polyclonal rabbit anti-S. neurona serum was added and themix was incubated 30 min. at 37° C. After incubation 150 μl washed(lysis buffer) GammaBind Plus Sepharose gel (Pharmacia) was added andincubated for 30 min. at 4° C. with shaking. The gel was washed bycentrifugation in a microfuge twice with lysis buffer and once withwater (to remove salt) and 135 μl 2DE sample buffer was added and heldat room temperature for 30 min. followed by centrifugation at 3000 g 30min. The supernate was removed to a new tube and centrifuged at fullspeed in a microfuge for 5 min. to remove any remaining gel.

A pH 3-10 IEF (Bio-Rad) strip was rehydrated overnight with 125 μl ofsample followed by isoelectric focusing with a step gradient: 200 V(0.01H), 3500V(1.5H), 3500V(2H). Strips were equilibrated in SDS buffer(2% SDS, glycerol, BME, 6M urea) for 15 min at room temperature,separated on 4-12% Bis-Tris gels and blotted to a PVDF membrane.

The approximate isoelectric point (pI) of S. neurona proteins separatedby 2D electrophoresis was determined from comparison with carbamylatedprotein standards. The standards were amylogulcosidase, 89 and 70 kDa,pI 3.8; ovalbumin 45 kDa, pI 5.1; carbonic anhydrase 29 kDa, pI 7.0;myoglobin 17 kDa, pI 7.6. The SnSAG1 reading frame codes for apolypeptide of 276 amino acids and a pI of 7.48. A spot that migrated atan apparent molecular weight of about 29 kDa and a pI of about 7.4 wasobserved on the blot.

CSF fluid from a horse with EPM was used to probe S. neurona antigensthat were separated in two-dimensional gels, blotted to PVDF membranes.The antigens of S. neurona recognized by CSF from a horse diagnosed withEPM were demonstrated. Those antigens identified by equine antisera werecompared with those detected when similar blots were probed from ahyperimmunized rabbit.

In an effort to elucidate the difference in antigens recognized byequine CSF and polyclonal rabbit anti-S neurona sera, furtherexperiments were done. Antigens were immunoprecipitated and separated bySDS-PAGE on a 4-12% Bis Tris gradient gel. The most outstandingobservation was that a horse with histologically confirmed case of EPMprecipitated the 30 kDa antigen that is detected by the rabbit antisera,but does not recognize this as the major antigen on a blot when probedwith equine CSF. Likewise, the rabbit antisera precipitated the 19 kDaantigen, but recognized the 30 kDa antigen as the predominant antigen.Although the rabbit antisera does not detect the 19/17 kDa antigen asdominant, when used to probe CSF immunoprecipitated antigens, the rabbitantisera was a better capture reagent than the equine CSF.

The polyclonal rabbit anti-S. neurona antibodies were then used toconcentrate the antigen by immunoprecipitation. The same blottedantigens detected with polyclonal rabbit anti-S. neurona antisera. Thehorse CSF antibodies percipitated antigens that separated atapproximately 28 kDa, however, the major antigens recognized by thehorse were at 19 kDa. The rabbit recognized major antigens at 29 kDa.

Example 7 Production of Anti-SnSAG-1 Antibodies

A mouse was immunized with S. neurona whole cell antigen and respondedwith an ELISA titer of greater than 1:8,000 to S. neurona culturedmerozoites. Antibodies to whole mouse immunoglobulin molecule serumlevels showed a good response to S. neurona but not host cells. Theantibodies were determined to be predominantly IgG. No appreciable titerto host cells was observed before or after immunization.

A hybridoma clone termed 1631 that secretes an anti-SnSAG-1 monoclonalantibody was produced by immunizing a mouse with whole S. neuronaparasites followed by routine fusion with myeloma cells. A well (1631)from the 96 well plate was tested and found to contain a monoclonalantibody (isotype IgG₁) to S. neurona. The cells were subjected tocloning by limiting dilution and transferred to a 24 well plate andcultured. This culture supernatant of 1631 reacted by ELISA at greaterthan 1:16,000 and immunoblotted whole cell S. neurona antigens to asingle band but not to host cells. Analysis of the supernatants fromthis culture indicated that it contained a monoclonal antibody thatspecifically bound (a) a 29 kDa band of blotted S. neurona antigens, and(b) the surface of whole formalin-fixed or methanol-fixed parasites byIFA and immunogold labeling EM. Epitopes were not removed by periodatetreatment as indicated by ELISA and immunogold labeling EM. The 1631antibody did not react to host cells, Neospora or T. gondii byimmunoblot. The 1631 antibody was used in an ELISA to determinecross-reactivity with two non-S. neurona Apicomplexan parasites known toinfect horses. The data indicated that 1631 does not bind Neosporahughesi merozoites, Toxoplasma gondii tachyzoites, or host cells.

The 1631 antibody preparation reacted with live cells or formalin fixedS. neurona but not host cells. Similar results were obtained withmethanol fixed parasites. The phase contrast image of the same fielddemonstrated that there was no binding to host cells. The controls forthis experiment, pre-immune mouse serum and isotype control, also showedno binding to S. neurona by immunofluorescence assay. Post-embeddingimmunogold labeling of S. neurona cultured merozoites using 1631antibodies also showed that the surface of the parasite was labeled.Controls included a mouse monoclonal isotype control. The clonedhybridoma 1631 was used to label the surface of S. neurona culturedmerozoites by post-embedding immunogold labeling.

Example 8 ELISA Protocols

Standard ELISA protocols were adapted for use to detect the presence ofS. neurona in a sample. Briefly, Nunc Maxisorp plates were prepared bythe addition of 50 μl S. neurona antigen or host cell antigen diluted to10 μg/ml in carbonate-bicarbonate buffer and incubate overnight at 4° C.The plates were washed 4 times and blocked in blocking buffer, 1% bovineserum albumin in 1×PBST (1×PBS, 0.05 Tween 20, 1% sodium azide) andincubated for 60 min. at room temperature. One hundred microliters ofprimary antibody at several dilutions (1631 mAb, mouse pre-immune serum,IgG, 1% BSA without serum, or media) was incubated for 1 hour at roomtemperature followed by four washes in blocking buffer. A commercialrabbit anti-mouse whole molecule (Sigma A 1902), goat anti-mouse IgG γchain specific (Sigma A3438), or goat anti-mouse IgM μ chain specific(Sigma A 9688) alkaline phosphatase conjugated secondary antibody wasadded and incubated for 60 min., washed three times with PBST followedby the addition of para-nitrophenyl phosphate. The OD was measured andrecorded at 492 nm at 30 and 60 min to provide results.

Example 9 Probing Clinical Tissues for the Identification of S. neurona

Rabbit anti-S. neurona antisera and 1631 monoclonal antibodies were usedto probe clinical tissues for the identification of S. neurona. In oneinstance, the 1631 antibody was used to detect S. neurona in the spinalcord of a horse with clinical signs of EPM. The hyperimmune rabbitantisera also identified merozoites in clinical tissue. Both reagentsclearly identified parasites in host tissues that were otherwiseidentified ambiguously when observed by hemosin and eosin stain.

The 1631 antibody was examined in a competitive ELISA to determine theuse of this assay in the measurement of antibodies in clinical samples.The assay used 1631 in a competitive ELISA that measured the percent ofinhibition of 1 microgram/ml of 1631 by equine serum and CSF. Althoughserum did competitively inhibit binding, CSF did not. The media used toprepare monoclonal antibodies was also found to compete for the antibodybinding site.

Example 10A Cloning and Expression of cDNAs Encoding Surface Antigens ofS. neurona

S. neurona merozoites were cultured as previously described. Ellison, S.P. et al., 2000, Vet. Parasitol., 1982:1-11. In this example, merozoitesat twelve days post infection were released with calcium ionophore andharvested in Hanks buffered salt solution. Washed merozoites were addedto RNAzol as per manufacturer recommendations. The total RNA wasseparated on a denaturing formamide gel to evaluate the amount of hostcell contamination. Vercammen, M. et al., 1998 Parasit. Immnol.,20(1):37-47. The separation of polyadenylated RNA (polyA RNA) from totalRNA was achieved by the use of magnetic beads as per manufacturerrecommendations.

Two methods were used to construct a cDNA expression library. The methodof Froussard was used to produce a cDNA library in the lambda vector UniZap XR. Froussard, Nucleic Acids Research, 20: 2900, 1992. First strandsynthesis was prepared using a 26 nucleotide primer containing a randomhexameter at the 3′ end (universal primer -dN6;5′-GCCGGAGCTCTGCAGAATTCNNNNN-3′) (SEQ ID NO:4). S. neurona poly A RNAwas suspended in 6 ml of distilled water, heated to 65° C. for 5 min,rapidly cooled on ice and reverse transcribed after addition of 0.5 ml(20 U) RNAsin, 1.25 ml 10×reverse transcription buffer (500 mM Tris-HCl,pH 8 at 43° C., 800 mM NaCl, 80 mM MgCl₂, 50 mM DTT), 1.25 ml dNTP (10mM), 1.5 ml universal primer-dN6 (0.1 mg/ml), 2 ml (16 U) AMU reversetranscriptase. Incubation was at 43° C. for 1 hour. The reaction wasthen boiled for 2 min. and rapidly cooled on ice. For second strand cDNAsynthesis the following components were added: 24.25 ml dH₂O, 10 ml5×Klenow buffer, 1.25 ml 5 methyl dCTP (100 mM), and 2 ml Klenowfragment (8 U). After 30 minutes incubation at 37° C., the sample waspurified on a chromaspin-400 column (Clontech) to remove the universalprimer d-N6 (sample 511). One microliter of the randomly synthesizeddouble stranded cDNA population was amplified in the presence of 1 mMuniversal primer in a 50 ml reaction mix containing 50 mM KCl, 1.5 mMMgCl₂, 0.01% gelatin, 500 mM each dNTP and 1.5 U TAQ polymerase. Thesamples were subjected to 40 cycles of amplification: 94° C. 1 min., 55°C. 1 min., and 72° C. 3 min. Final amplification products analyzed on a0.8% agarose gel. The amplified cDNA fragments were ligated to EcoR1adapters and separated on a drip column containing Sepharose CL-2B gelfiltration medium. The size fractionated cDNA was precipitated andligated to the Uni Zap XR vector. The lambda library was packaged into ahigh efficiency Gigapack III gold packaging extract and plated on the E.coli cell line XL1-Blue MRF-.

A second cDNA expression library was constructed in the lambda vectorUni Zap XR (Stratagene) as per the manufacturer's recommendations. PolyARNA was converted into double stranded cDNA using the ZAP cDNA synthesiskit and subsequently ligated with Novagen PST Blue perfectly bluntcloning kit (PST Blue-1 vector has EcoR1 sites flanking cloning sites).After in vitro packaging and transfection of host cells (XL1-Blue MRF-)the primary library was amplified to yield a high titer phage stock.

To screen for recombinant phage clones expressing a putative surfaceantigen of Sarcocystis neurona both a monoclonal antibody (antibody from1631 culture supernatant) and polyclonal rabbit anti-S. neuronaantibodies were used. The library was also screened with a secondmonoclonal antibody, 2A7 (a gift of Antoinette Marsh). Analysis of thisantibody indicated that it specifically bound (a) the surface of S.neurona as determined by by IFA, and (b) a 29 kDa antigen of blotted S.neurona parasites. The 2A7 antibody was also subjected to IFA,immunogold labeling electron microscopy, and western blotting for therecombinant work.

Polyclonal anti-S. neurona rabbit antibodies were produced by LampireLaboratories with whole antigen that had been fresh frozen and shipped.Screening was performed according to standard procedures. Primaryantibodies were diluted 1:500. Alkaline phosphatase-conjugated goatanti-mouse IgG or goat anti-rabbit IgG were used as secondary antibodies(dilution 1:3000). Positively reacting phage plaques were isolated andsubjected to 3 additional rounds of screening. Positive phage cloneswere converted into pBluescript SK (−) phagemids by means of in vivosubcloning.

The in vivo excision protocol (Stratagene) was followed. Briefly, 1 mlof the amplified library was diluted by addition of SM buffer to give400 pfu/ml. Overnight cultures of XL1-Blue MRF- and SOLR, supplementedwith 0.2% (w/v) maltose and 10 mM MgSO₄ were centrifuged and the mediaremoved. The cells were resuspended to an OD₆₀₀ of 1 in 10 mM MgSO₄. Twohundred microliters of XL1-Blue MRF- were added to 250 ml (1×10⁵) phageand 1 ml Ex Assist helper phage (1×10⁶ pfu/ml) and incubated at 37° C.for 15 minutes in a Falcon 2059 polypropylene tube. Three ml LB brothwas then added and the tube incubated 2 hours at 37° C. with shaking.The tube was then heated to 70° C. for 20 minutes and centrifuged at1000×g for 15 min. The supernatant containing the excised pBluescriptphagmid was plated by adding 200 ml SOLR cells to 100 ml phagesupernatant, and the mixture was incubated for 15 min at 37° C. Twohundred ml of the cell mixture was plated on LB-ampicillin agar plates(50 mg/ml) and incubated overnight.

Additionally, 96 random phagmids were selected, grown overnight, andplasmids isolated by standard mini-prep procedures. The isolatedplasmids were sequenced and one candidate clone, C10, was selected basedon BLAST analysis. Altschul, S. F. et al., 1997 Nuc. Acids Res.,25:3389-3402. The cDNA library was screened by hybridization with adigoxigenin-labeled probe (EcoR1/Xho1), a fragment from clone CIO.Sequence analysis of one clone revealed a full-length copy of the genethat contained the C10 fragment, a gene with significant similarity tothe major surface antigen of S. muris (smMSA1).

PCR primers 5′ GGGGGGATCCGATGACGAGGGCGGGTGCTGCTG (SEQ ID NO:5) and 3′GGGGGATCCTTAGCAAAAGTGCAAGAAAGCG (SEQ ID NO:6) were designed by adding aBamH1 nucleotide sequence to the sequence encoding the 5′ and 3′flanking ends of the open reading frame of the full gene encoding theputative surface antigen of S. neurona. A standard 50 ml PCR reactionusing 1 ml second strand synthesis reaction as template was used. Thesingle amplification produced by PCR was isolated by electrophoresis andcut out of a low melting agarose gel followed by TOPO (Invitrogen)cloning. One colony was selected. The size of the insert was determinedby electrophoresis and the sequence was confirmed by sequence analysis.The DNA was then cloned into the BamH1 site of pBluescript, transfectedinto XL1-Blue MRF—, and color selected. The DNA from the positive colonywas sub-cloned into an expression vector for protein expression.Expression of fusion protein-The cDNA insert from clone 1631-5 andC10-511 was subcloned into the BamH1 site of the expression vectorpet14b. This vector allows the expression of the target gene in E. coliunder the control of strong bacteriophage T7 promoter. The gene wasestablished in the non-expression host XL1-B. Plasmids were thentransferred into the expression host p21 that contains a chromosomalcopy of the T7 RNA polymerase gene under lacUV5 control. Expression wasinduced by the addition of 0.4 mM IPTG. Additionally, this vectorcontained a HIS “tag” which facilitates detection and purification ofthe target protein. The gene for ampicillin resistance, (b-lactimase),is contained in the plasmid in the same orientation as the target gene.Forty colonies of bacteria containing the C10-511 gene that grew onampicillin containing LB plates after overnight incubation were selectedand mini-preps prepared. DNA digests using Sma1 and BamH1 were used toselect recombinants in the correct orientation. Seven recombinants inthe correct orientation were identified. The 1631 recombinants did notallow selection of the correct orientation by DNA digestion. Tenrecombinants were selected from ampicillin containing LB plates afterovernight incubation. The presence of the 1631 gene was determined byPCR amplification using primers that were developed to add BamH1sequence to the gene for cloning into p21 and primers used in sequencingthe insert. The correct orientation was selected by the size of theresulting PCR product.

The ATG codon at the beginning of the reading frame is preceded by twoin-frame termination codons at positions −39 and −66 in the 5′ upstreamregion. The 828 bp reading frame codes for a polypeptide of 276 aminoacids with a calculated molecular mass of 28,328 is daltons and anisoelectric point (pI) of 7.48. This sequence appears to encode amembrane protein with a cleavable signal peptide and one transmembranespanning region near its C terminus. McGeoch 1985 1949/id; Hartmann,Rapoport, et al. 1989 1950/id. It has a potential cleavage site at Ala255 suggesting a possible GPI anchor at this position. Eisenhaber, Bork,et al. 1999 1951/id. The sequence has 12 cysteine residues, 10 of whichare conserved when compared with SmSAG1, and 9 are conserved when thecomparison is extended to N. caninum SAG1 (NcSAG1, GenBank No.AF132217). Examination of the genomic DNA encoding SnSAG-1 by PCRindicated that the genomic sequence was slightly larger than that of thecDNA. Sequence analysis indicated the presence of a single 128 bp intronlocated at position 406/407 in the coding region of the mature mRNA.Over-expression of SnSAG-1 in E. coli using pET14b produced arecombinant protein, with an N-terminal sequence containing 6 Hisresidues, that is slightly larger than the native antigen 29 kDa.

Bacteria carrying the recombinant plasmid for C10-511 were grown to anA₆₀₀ of 0.6 in a 50 ml culture. Twenty ml of culture was added to 1liter of LB-ampicillin broth (50 mg/ml) and grown to an A₆₀₀ of 0.6.Isopropl thyogalactoside (IPTG) was then added to a final concentrationof 0.4 mM and incubation was continued for 2 hours. After harvesting thecells, the fusion protein was detected by immunoblot using anti HIS-tagmonoclonal antibody.

Example 10B Cloning and Expression of cDNAs Encoding Surface Antigens ofS. neurona

S. neurona merozoites were cultured as previously described. Ellison, S.P. et al., 2000, Vet. Parasitol., 1982:1-11. In this example, merozoitesat twelve days post infection were released with calcium ionophore andharvested in Hanks buffered salt solution. Washed merozoites were addedto RNAzol as per manufacturer recommendations. The total RNA wasseparated on a denaturing formamide gel to evaluate the amount of hostcell contamination. Vercammen, M. et al., 1998 Parasit. Immnol.,20(1):37-47. The separation of polyadenylated RNA (polyA RNA) from totalRNA was achieved by the use of magnetic beads as per manufacturerrecommendations.

A cDNA expression library was constructed in the lambda vector Uni ZapXR (Stratagene) as per the manufacturer's recommendations. An oligo dTprimer with a linker containing an XhoI restriction site was used toprime for 1st strand cDNA synthesis using MMLV reverse transcriptase butincluding 5-methyl dCTP to hemi-methylate the transcripts. This protectsthe cDNA from later XhoI digestion while leaving the unmethylated linkerprimer open to digestion. After 1st strand synthesis, 2nd strandsynthesis was performed by treating with RNase H and DNA polymerase I.The ends of the double stranded cDNA molecules produced in this reactionwere polished using Pfu DNA polymerase, ligated to EcoRI adaptors andphosphorylated. XhoI digestion was performed preparing the transcriptsfor directional cloning into the Unizap XR vector and size fractionatedover a Sepharose CL-2B column. These cDNA inserts were ligated into theUnizap XR cloning vector and the ligation mixture was packaged intofunctional lambda phage partices using the Gigapack Gold packagingextract (Stratagene, Inc.).

The in vivo excision protocol (Stratagene) was followed to provideexcised phagemids from the primary UniZAP library. Ninety-six of theserandomly selected phagmids were grown overnight, and plasmids isolatedby standard mini-prep procedures. The isolated plasmids were sequencedand the data were analyzed by BLAST comparison against the nr databaseat NCBI (Altschul, S. F. et al., 1997 Nuc. Acids Res., 25:3389-3402).Two candidate clones, C11 and F03, were identified by sequencesimilarity with the major surface antigen of S. muris (smMSA1). The cDNAlibrary was screened by hybridization with ³²P-labeled insert from theC11 clone yielding a large number of phage clones hybridizing with thisprobe. Six of these positive clones were selected and sequenced yieldingthe complete sequence of the coding region. PCR primers JNB162-(ACGAGGATCCGATGACGAGGGCGGTGCTGCTG) (SEQ ID NO:7) and JNB163-(ACGAGGATCCCACGGCAGGATTAGCAAAAGTGC) (SEQ ID NO:8) were designed toadd a BamH1 recognition sequence for cloning into the pET14b expressionvector (Novagen). The pool of cDNA prepared from merozoite mRNA was usedas template. The single amplification produced by PCR was isolated byelectrophoresis and cut out of a low melting agarose gel followed byTOPO (Invitrogen) cloning. One colony was selected. The size of theinsert was determined by electrophoresis and the sequence was confirmedby sequence analysis. The DNA from the positive colony was sub-clonedinto pET14b for protein expression. The insert was released by BamHIdigestion, purified by low melting agarose gel electrophoresis andligated into the pET14b vector. Orientation was determined byrestriction digestion with SmaI. Seven recombinants in the correctorientation were identified.

In the largest cDNA clone the ATG codon at the beginning of the readingframe is preceded by two in-frame termination codons at positions −39and −66 in the 5′ upstream region. The 828 bp reading frame codes for apolypeptide of 276 amino acids with a calculated molecular mass of28,328 daltons and an isoelectric point (pI) of 7.48. This sequenceappears to encode a membrane protein with a cleavable signal peptide andone transmembrane spanning region near its C terminus. McGeoch 19851949/id; Hartmann, Rapoport, et al. 1989 1950/id. It has a potentialcleavage site at Ala 255 suggesting a possible GPI anchor at thisposition. Eisenhaber, Bork, et al. 1999 1951/id. The sequence has 12cysteine residues, 10 of which are conserved when compared with SmSAG1,and 9 are conserved when the comparison is extended to N. caninum SAG1(NcSAG1, GenBank No. AF132217). Examination of the genomic DNA encodingSnSAG-1 by PCR indicated that the genomic sequence was slightly largerthan that of the cDNA. Sequence analysis indicated the presence of asingle 128 bp intron located at position 406/407 in the coding region ofthe mature mRNA. Over-expression of SnSAG-1 in E. coli using pET14bproduced a recombinant protein, with an N-terminal sequence containing 6His residues, that is slightly larger than the native antigen 29 kDa.

Bacteria carrying the recombinant plasmid for C10-511 were grown to anA₆₀₀ of 0.6 in a 50 ml culture. Twenty ml of culture was added to 1liter of LB-ampicillin broth (50 mg/ml) and grown to an A₆₀₀ of 0.6.Isopropl thyogalactoside (IPTG) was then added to a final concentrationof 0.4 mM and incubation was continued for 2 hours. After harvesting thecells, the fusion protein was detected by immunoblot using anti HIS-tagmonoclonal antibody.

Example 11 Southern and Northern Blot Hybridization

S. neurona merozoites were harvested from 12 days post-infectioncultures and the total DNA was extracted using standard procedures.Briefly, the whole cells were pelleted in PBS. The pellet was suspendedin lysis buffer (1M Tris, pH 8, 0.5M EDTA, pH 8, 5 M LiC1, Triton X100). An equal volume of phenol: chloroform: isoamyl alcohol (1:1:25)was added and mixed by inversion for 2 minutes. The suspension wascentrifuged at 10,000 rpm in a microfuge at 4° C. for five minutes toseparate the liquid phases. The aqueous phase was removed to a new tubeand two and one half volumes 100% EtOH was added and incubated for 20min at −20° C. The precipitated DNA was pelleted by centrifugation at14,000 rpm for 20 min at 4° C. The pellet was washed in 80% EtOH, airdried and resuspended in 50 ml TE. DNA extracted from T. gondii andNeospora served as controls. DNA (5 mg) aliquots were digested withEcoR1 or BamH1 and electrophoresed on a 0.8% (w/v) agarose gel. Southernblotting was preformed by capillary transfer onto nylon membranesfollowing acid depurination, alkaline denaturing, and neutralizationsteps.

Hybridization probes were generated by PCR isolated recombinants. Theprimers: 5′ GGGGGGATCCGATGACGAGGGCGGGTGCTGCTG (SEQ ID NO:5) and 3′GGGGGATCCTTAGCAAAAGTGCAAGAAAGCG (SEQ ID NO:6) (in other experimentsprimers JNB 162 and JNB 163 were used) were used in a standard PCRreaction with one microliter of DNA from a clone that contained the openreading frame of the gene SnSAG-1. The resulting single PCR band wasdiluted and an aliquot labeled with ³²P by random priming. This probewas used in hybridization to DNA and mRNA from whole S. neurona, T.gondii, Neospora, and S. falcatula. The blots were hybridized using1×SSC at 60° C. Bands were visualized using manufacturerrecommendations.

For Northern analysis, RNA was produced as described above and wasanalyzed by agarose/formaldehyde gel electrophoresis. The RNA, 1 μg, wastransferred to a nylon membrane. After pre-hybridization (2×SSCcontaining salmon sperm DNA) for two hours at 60° C., a probe preparedfrom the open reading frame of SnSAG-1 was ³²P labeled by the randomprimer method and added to the hybridization buffer for 24 hours at 60°C. Feinberg, A. P. and Vogelstein, B., 1984 Anal. Biochem., 137:266-267.The membrane was washed in 0.2×SSC, 0.2% SDS at 55° C. Autoradiographywas performed with enhancing screens for 8 days at −80° C. The probe wasalso used to hybridize blots of DNA from S. neurona, T. gondii, N.caninum, and S. falcatula. Bands were visualized by autoradiography.

Clones from a cDNA library constructed from the mRNA of S. neuronamerozoites cultured in vitro were randomly sequenced. Partial sequenceof one clone containing a 687 bp insert when translated into an aminoacid sequence had 32% identity to the major surface antigen of S. muris(SmMSA1). This partial clone was used as a probe to obtain a full-lengthcopy of the coding region of the gene. Screening the library byhybridization with this probe demonstrated that ˜10% of clones screenedbound this probe. Analysis of seven positive clones revealed that manyof the cDNA inserts were partial clones of similar length (˜0.83 kb),and they shared a common restriction map (data not shown). One largerclone had an extended 5′-untranslated region with a total insert size of1232 base pairs. The sequence of this longer clone contains a 5′noncoding region of 72 bp, a single long open reading frame of 828 bp,and a 333 bp 3′ nontranslated region. When the probe was hybridized to ablot of total RNA isolated from S. neurona, one extremely strong bandwas detected at 1.5 k. It thus appears that the cDNA clone sequencedcontains most of the major mRNA transcript. This probe also hybridizedto S. neurona DNA fragments but not to bovine host cell DNA. A blot ofEcoR1-cleaved DNA hybridized with this probe yields a single band atapproximately 1.2 kb.

Example 12 Preparation of rSnSAG-1

Four liters of induced cells were collected by centrifugation at 6000rpm in a JB6 rotor 1000×g at 4° C. for twenty minutes. Cells wereresuspended in 25 ml TN buffer (50 mM Tris, pH 7.4, 150 mM NaCl).Several methods were compared including freeze/thaw and sonication forthe release of inclusion bodies. A higher yield of recombinant proteinwas released when a French press was used to rupture the cells. Thecells were disrupted by passing through a French press twice at 2200PSI. One-fourth of the mixture was then layered over a 28 ml 27% sucrosecushion and centrifuged at 9000 rpm for 30 min at 10° C. using a SW41rotor at 7000×g in an ultracentrifuge. This step was repeated.

The resulting pellet was resuspended in one ml TN buffer, divided into250 ml aliquots in microfuge tubes and stored at −20° C. One aliquot wasmixed with 6M urea in 1×bind buffer (40 mM imidazole, 4M NaCl, 160 mMTris, pH 7.9) and stirred for 40 minutes at 4° C. The solution wascentrifuged at 9000 rpm for 30 minutes at 5° C. A His-Tag column wasprepared by washing 2 ml resin with three volumes distilled waterfollowed by six volumes 8×charge buffer (400 mM NiSO4) diluted to 1×with distilled water. The column was washed with three volumes 1×bindingbuffer containing 6M urea. The sample was added and the column washedwith 10 volumes 1×bind buffer containing 6M urea, 6 volumes 1×washbuffer made by diluting 8× wash buffer (480 mM imidazole, 4 M NaCl, 160mMTris pH 7.9) with distilled water. The sample was eluted using 1×stripbuffer diluted from a 4×stock (400 mM EDTA, 2 M NaCl, 80 mM Tris pH7.9). The sample was then dialyzed to remove urea and salts using 20 mMTris pH 8 with 1% Triton X. The buffer was changed three times over 24hours.

Over-expression of SnMSA-1 in E. coli using pET 14b produced arecombinant protein of 29 kDa, pI 7.3, with an N-terminal extensioncontaining 6 His residues and a thrombin cleavage site. Purification ofthe recombinant protein was achieved using a His-tag column. The His-tagwas confirmed by N-terminal sequencing and was recognized on immunoblotsby His-tag monoclonal antibody. Carbamylated protein standards were usedto determine the pI of rSnMSA-1 focused on IPG strips at pI 7.3.Thrombin cleavage of the recombinant protein to release the His-tagyielded rSnMSA-1 that was recognized on immunoblots by mAbs 1631, 2A7,polyclonal monospecific mouse anti-rSnMSA1, equine serum and CSF, butnot serum from European horses or monoclonal isotype controls. Cleavageof the His-tag from the recombinant protein decreased the apparentmolecular weight slightly as detected by SDS-PAGE analysis. Monospecificanti-rSnMSA-1antisera bound native S. neurona surface antigens by IFA,post embedding immunogold labeling and ELISA confirming the identity ofthis clone as a surface antigen of this parasite.

Example 13 Capture ELISA was used to Quantitate S. neurona Antigens

Antibodies raised against S. neurona were used to quantitate rSnSAG-1that bound in a capture ELISA. The capture ELISA using rSnSAG-1 servedas a standard to measure the amount of SnMSA-1 used in preparativesamples for immunoblot. First, recombinant protein was used in an ELISAto titer purified IgG from polyclonal rabbit anti-S. neurona antibodies.A second aliquot of purified antibody was biotinylated and used as areagent to detect antigen captured by antibodies, this reagent was alsoquantitated using against rSnSAG-1. Both protein G purified andbiotinylated protein G purified rabbit anti-S. neurona were used toconfigure a capture ELISA. 96 well plates were coated with protein Gpurified rabbit anti-S. neurons, followed by the addition of dilutedrSnSAG-1. After incubation and washing using standard conditionsbiotinylated protein G purified rabbit anti-S. neurona was added,incubated, and washed. The detection of the biotinylated antibodies wasaccomplished with the addition of alkaline phosphatase-Strepavadin(P-NPP,(Sigma N2765) and the absorbance read at 405 nm. Additionally,the purified rabbit anti-S. neurona was used as the capture antibody andmonoclonal antibodies directed against SnMSA-1 were used for detectionof protein captured. Monospecific anti-rSnMSA-1 antisera bound native S.neurona surface antigens by ELISA confirming the identity of this cloneas a surface antigen of this parasite.

Example 14 Localization of SnSAG-1 on Surface of S. neurona Merozoites

Standard IFA protocols were used, but fixation of the parasites varied.S. neurona was grown in BM cells on glass slides that were coated withcollagen followed by fixation in methanol or formalin. Parasites wereharvested from 150 cm² flasks and washed with PBS and used fresh orprepared by cytospin and fixed with methanol or formalin. Slides wereblocked with the application of 100 μl dilute goat serum for 30 min. at37° C. to block non-specific binding. Slides were washed in PBST bathfor 5 min. with three changes of buffer, drained of excess fluid andprimary antibody.

Monoclonal antibodies 1631 and 2A7 bound strongly to S. neuronamerozoites as viewed by immunofluorescence microscopy. Parasites wereexamined by transmission electron microscopy after immunogold labelingusing standard procedures used at the ICBR Electron Microscopy Core atthe University of Florida. Briefly, parasites were embedded in epoxy fortransmission electronmicroscopy. Grids containing thin sections wereincubated with mAb control sera, mAb 2A7, or mAb 1631 and incubated forone hour at room temperature. Secondary anti-mouse antibodies conjugatedto 15 nm gold beads were applied. Stained sections were subsequentlyviewed by transmission electron microscopy. Gold-labeled, anti-mouseantibodies bound the surface of the embedded parasites treated witheither mAb 2A7 or mAb 1631.

Example 15 Immunoblot Analysis

Monoclonal antibodies 1631 and 2A7 bound a 29 kDa antigen on immunoblotsof SDS-PAGE separated native antigens derived from cultured S. neuronamerozoites. The immunodominance of antigen(s) at ˜29-30 kDa in a mouseimmunized with merozoites suspended in adjuvant was evident. Binding ofmAb2A7 and mAb1631 to an antigen at 29 kDa was evident. When the antigenpreparation is reduced by heating in the presence of β-mercaptoethanol,binding of both monoclonal antibodies was greatly reduced or abolished.Neither of these monoclonal antibodies bound to host cell antigens norto N. caninum or T. gondii tachyzoite antigens on immunoblots.

The rSnSAG-1 was recognized on immunoblots by His-tag monoclonalantibody, mAb 2A7, CSF from a horse with clinical EPM, but not by serumfrom European horses never exposed to S. neurona. Monoclonal antibodies2A7 and 1631 bind to epitopes on rSnSAG-1 that are sensitive toreduction with sulfhydryl reducing agents, but amounts ofβ-mercaptoethanol twice normal are necessary to eliminate the epitopefrom rSnSAG-1 on western blots. IgG1 control antibody does not bind torSnSAG-1 non-reduced or reduced. Serum from a horse with clinical EPMbinds to rSnSAG-1 in a fashion similar to the mAbs 2A7 and 1631. Whenβ-mercaptoethanol is increased in the sample buffer from 5 to 10%, theamount of detectable rSnSAG-1 is greatly reduced.

Results of the above experiments determined that both monoclonalantibody 2A7 and the rabbit polyclonal antibody bound the recombinantprotein as well as a 29 kDa protein of the cultured parasite. Therecombinant protein was detected as a 30 kDa band when the His-tagremained on the protein. In parallel experiments, equine CSF from ahistopathologically confirmed case of EPM, but not European equine sera,was also shown to bind the 29 kDa band.

Protein samples including merozoites and rSnSAG-1 were suspended in4×sodium dodecyl sulfate polyacryamide gel electrophoresis (SDS-PAGE)loading buffer without reducing agents (1.09 M glycerol, 141 mM Trisbase, 106 mM Tris HCL, 73 mM lithium dodecyl sulfate, 0.51 mM EDTA, 0.22mM Serva Blue G250, 0.175 mM phenol red, pH 8.5). After boiling, sampleswere loaded onto 4-12% New Page Bis Tris gels. For Western blotanalysis, proteins were transferred onto PVDF membranes andimmunodetection as described below. Briefly, 10 mg of purified parasitesor protein preparations were electrophoresed on 4-Bis-Tris 12% reducingor non-reducing gels and transferred to PVDF membranes. PVDF membraneswere blocked for one hour in PBS with 5% non-fat dry milk and 2% Tween20.

Rabbit anti-S. neurona polyclonal (polyclonal antibodies were made byimmunizing a rabbit with an antigen prearation made from the UCD 1isolate) or specific monoclonal antibody (2A7, a gift of AntoinetteMarsh) was used as a primary antibody. Primary antibody was diluted inblocking buffer at 1:500 was incubated on a rotary shaker for two hoursat room temperature. Membranes were washed for one hour with threechanges of blocking buffer. Alkaline phosphatase-conjugated secondaryantibody was diluted to manufacturer's recommendations (1:15,000) andincubated at room temperature on a rotary shaker for one hours.Membranes were washed for one half hour with three changes of blockingbuffer. Chromogenic detection of antibody was preformed as themanufacturer recommended (Invitrogen Westernbreeze chromogenic kit #WB7103 and WB7105). Pooled sera from 6 European horses that were neverexposed to S. neurona was used as a negative control. Serum and cerebralspinal fluid from an S. neurona-infected equine were obtained from thehorse from which the UCD 1 isolate was cultured. Marsh, A. E. et al.,JAVMA, 209(11)1907-1913.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 8 <210> SEQ ID NO 1 <211> LENGTH: 276<212> TYPE: PRT <213> ORGANISM: Sarcocystis neurona <400> SEQUENCE: 1Met Thr Arg Ala Val Leu Leu Thr Phe Leu Th #r Leu Cys Ser Ala Arg1               5    #                10   #                15Val Ser Leu Val Arg Ala Gly Ala Pro Pro Gl #n Ala Thr Cys Ala Asn            20       #            25       #            30Gly Glu Thr Thr Val Thr Lys Leu Gly Ser Se #r Gly Ala Leu Arg Ile        35           #        40           #        45His Cys Pro Asn Asn Phe Arg Leu Ala Pro Ar #g Ala Gly Asn Asp Ala    50               #    55               #    60Gly Gln Met Gln Val Tyr Ala Thr Ala Val Al #a Glu Asn Pro Val Asn65                   #70                   #75                   #80Ile Arg Asp Val Leu Pro Gly Ala Ser Tyr Le #u Ser Val Gln Asn Val                85   #                90   #                95Pro Thr Leu Thr Val Pro Gln Leu Pro Ala Ly #s Ala Thr Ser Val Phe            100       #           105       #           110Phe His Cys Gln Gln Gln Pro Asp Asn Gln Cy #s Phe Ile Gln Val Glu        115           #       120           #       125Val Ala Pro Ala Pro Arg Leu Gly Pro Asn Th #r Cys Ala Ala Leu Gln    130               #   135               #   140Ser Thr Ile Ala Phe Glu Val Gln Gln Ala As #n Glu Thr Ala Val Phe145                 1 #50                 1 #55                 1 #60Ser Cys Gly Glu Gly Leu Ala Val Phe Pro Gl #n Gly Ser Lys Ala Leu                165   #               170   #               175Asp Glu Ala Cys Ser Lys Glu Gln Ala Leu Pr #o Ser Gly Ala Ala Leu            180       #           185       #           190Ala Pro Lys Asp Gly Gly Leu His Leu Gly Ph #e Pro Gln Leu Pro Gln        195           #       200           #       205Gln Ala Met Lys Ile Cys Tyr Ile Cys Thr As #n Gly Gly Val Gln Ala    210               #   215               #   220Glu Ala Ala Gln Arg Cys Glu Val Arg Ile Se #r Val Ala Ala Asn Pro225                 2 #30                 2 #35                 2 #40Asp Ser Gly Val Pro Gly Ala Asn Gly Ala Al #a Ser Leu Gly Ala Ala                245   #               250   #               255Ala Arg Ser Ala Ser Ala Leu Gly Leu Ala Le #u Val Ala Gly Ala Phe            260       #           265       #           270Leu His Phe Cys         275 <210> SEQ ID NO 2 <211> LENGTH: 1132<212> TYPE: DNA <213> ORGANISM: Sarcocystis neurona <400> SEQUENCE: 2gggaggtaag tgttggcggt aatgctgcat cattagggtc agacacgctg tc#catctgtc     60attctcgcca gaatgacgag ggcggtgctg ctgacgtttc tgacactctg ct#ccgccaga    120gtgtcccttg tgagggccgg agcgccgcct caagcacgtg cgccaatggc ga#aacgactg    180ttactaagct cggcagctct ggcgcactac gaatccactg cccaaataat tt#tcgactcg    240cgccccgggc tgggaatgac gccggtcaga tgcaggtcta tgcactgcgg tt#gctgagaa    300tcctgtaaac atacgagacg tcctgcccgg cgcatcttac ctctctgtac ag#aacgtccc    360gagggtcacc gtcccgcaat tgcccgccaa agctacgagc gtcttttttc ac#tgccagca    420gcaacccgac aaccaatgct tcatccagtc agctgcggcg agggacttgc tg#tgttcccg    480caaggtagca aagcgttgga tgaagcctgc tccaaagagc aggccctacc ca#gtggcgcc    540gctttagctc caaaggatgg tgggctccac cttggttttc ctcagcttcc tc#agcaggct    600atgaagattt gctatatttg tacgaatggt ggtgtgcagg cagaggcggc cc#aacggtgt    660gaggttcgca tctccgtcgc agcgaaccca gacggaagcg ttccaggggc ta#acggagcc    720gcctctctag gagctgccgc acgcagcgcc tctgcgttag ggttggctct cg#ttgcaggc    780gctttcttgc acttttgcta atcctgccgt gtagcgtctc tggtggcccg cc#ccacagat    840cctggttatt cccacagcgt gtagcgtctc tggtggcccg ccccacagat cc#tggttacg    900cgttcagtaa cgtgcctact gttccaaaac gggaaaatcc gaagatgcaa aa#ttcatccg    960gtgcagcgtc ccatgtgttc agttacgact ggacgagtgt agtgagatgg tt#ttacatcc   1020attcgcagtg cagaggcgtg ggctcgcata tttttttttt gtagtgtgcc gt#tgtagatc   1080cagcaagtta aatatgttag tcattttgag cgcctgttcc acgtaggcgg ct#           1132 <210> SEQ ID NO 3 <211> LENGTH: 729 <212> TYPE: DNA<213> ORGANISM: Sarcocystis neurona <400> SEQUENCE: 3atgacgaggg cggtgctgct gacgtttctg acactctgct ccgccagagt gt#cccttgtg     60agggccggag cgccgcctca agcacgtgcg ccaatggcga aacgactgtt ac#taagctcg    120gcagctctgg cgcactacga atccactgcc caaataattt tcgactcgcg cc#ccgggctg    180ggaatgacgc cggtcagatg caggtctatg cactgcggtt gctgagaatc ct#gtaaacat    240acgagacgtc ctgcccggcg catcttacct ctctgtacag aacgtcccga gg#gtcaccgt    300cccgcaattg cccgccaaag ctacgagcgt cttttttcac tgccagcagc aa#cccgacaa    360ccaatgcttc atccagtcag ctgcggcgag ggacttgctg tgttcccgca ag#gtagcaaa    420gcgttggatg aagcctgctc caaagagcag gccctaccca gtggcgccgc tt#tagctcca    480aaggatggtg ggctccacct tggttttcct cagcttcctc agcaggctat ga#agatttgc    540tatatttgta cgaatggtgg tgtgcaggca gaggcggccc aacggtgtga gg#ttcgcatc    600tccgtcgcag cgaacccaga cggaagcgtt ccaggggcta acggagccgc ct#ctctagga    660gctgccgcac gcagcgcctc tgcgttaggg ttggctctcg ttgcaggcgc tt#tcttgcac    720 ttttgctaa                 #                  #                   #        729 <210> SEQ ID NO 4 <211> LENGTH: 25<212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE:<223> OTHER INFORMATION: universal primer <221> NAME/KEY: misc_feature<222> LOCATION: (21)..(25) <223> OTHER INFORMATION: n <400> SEQUENCE: 4gccggagctc tgcagaattc nnnnn           #                  #               25 <210> SEQ ID NO 5 <211> LENGTH: 33 <212> TYPE: DNA<213> ORGANISM: Sarcocystis neurona <400> SEQUENCE: 5ggggggatcc gatgacgagg gcgggtgctg ctg        #                  #         33 <210> SEQ ID NO 6 <211> LENGTH: 31 <212> TYPE: DNA<213> ORGANISM: Sarcocystis neurona <400> SEQUENCE: 6gggggatcct tagcaaaagt gcaagaaagc g         #                  #          31 <210> SEQ ID NO 7 <211> LENGTH: 32 <212> TYPE: DNA<213> ORGANISM: Sarcocystis neurona <400> SEQUENCE: 7acgaggatcc gatgacgagg gcggtgctgc tg        #                  #          32 <210> SEQ ID NO 8 <211> LENGTH: 33 <212> TYPE: DNA<213> ORGANISM: Sarcocystis neurona <400> SEQUENCE: 8acgaggatcc cacggcagga ttagcaaaag tgc        #                  #         33

What is claimed is:
 1. A composition for stimulating an immune responseagainst Sarcocystis neurona when administered to an animal, thecomposition comprising: (a) an isolated polypeptide comprising the amineacid sequence as set forth in SEQ ID NO:1; and (b) a pharmaceuticallyacceptable carrier.
 2. The composition of claim 1, wherein thecomposition comprises an adjuvant.
 3. The composition of claim 2,wherein the adjuvant comprises a substance selected from the groupconsisting of: an aluminum salt; an oil-in-water emulsion; a compositioncomprising saponin; a composition comprising a bacterial protein; and acytokine.
 4. A composition for detecting the presence of S. neurona in abiological sample comprising an isolated polypeptide as set forth in SEQID NO:1.
 5. The composition according to claim 4, wherein thepolypeptide is fused, in frame, to another polypeptide.
 6. Thecomposition according to claim 4, wherein the polypeptide is arecombinant protein.
 7. An isolated polypeptide comprising the aminoacid sequence as set forth in SEQ ID NO:1.
 8. The isolated polypeptideof claim 7, wherein the polypeptide is fused, in frame, to anotherpolypeptide.
 9. The isolated polypeptide of claim 7, wherein thepolypeptide is a recombinant protein.
 10. The composition according toclaim 4, further comprising a pharmaceutically acceptable carrier. 11.The composition according to claim 1, wherein said polypeptide is fused,in frame, to another polypeptide.